Membrane tension profiles revealed cell edges uncoupling during Glioblastoma linear motility

Glioblastoma cells exploit blood vessels and neuronal axons to invade the brain. At the microscale level, the geometry and confinement of these physiological tracks impact cell mechanics, particularly the plasma membrane tension and, in turn, cell migration. In the past, several studies widely explored the participation of plasma membrane tension in cellular physiological processes, such as cell spreading, polarisation, phagocytosis, and membrane trafficking, emphasising tension contribution in counteracting actin cytoskeleton remodelling. However, the role of membrane tension as a mechanical regulator of cell motility is controversial, and the scientific community still discusses whether it can regulate cell behaviour locally or globally. Discrepancies may arise from the use of different cell types and tools for probing membrane tension. Various membrane-pulling assays have been used to investigate membrane tension, and variation has been recorded during cell front protrusion. Unfortunately, current approaches are unsuitable for studying membrane tension evolution at the whole cell level. Moreover, despite the impact of cholesterol on plasma membrane tension being well consolidated, a significant number of studies focused on lipid metabolism to curb Glioblastoma progression, disregarding the mechanical effect on Glioblastoma cell invasion. This PhD project proposed a novel in-vitro vessel-mimicking approach for studying membrane tension's spatiotemporal evolution in invasive Glioblastoma cells. A fluorescent membrane dye (FLIPPER-TR) has been used to record membrane tension's evolution by analysing the fluorescence decay along the whole cell. Deep-UV micropatterning has been used as a vessel-mimicking approach to create linear cues with micro-scaled dimensionalities, similar to brain capillaries, and resemble brain vasculature's geometry and constrain. Cell-instructive patterned surfaces were coated with laminin, the most representative matrix protein of the abluminal surface of brain vasculature, to mimic the perivascular niche composition. Combining the micropatterning technique with the Fluorescence-Lifetime Imaging Microscopy (FLIM) of FLIPPER-TR probe, we have integrated subcellular tension variations with global cell motility. Results showed different membrane tension patterns at the cell front and rear, separated by a low-tension zone, which buffers tension transmission and sustains the independent behaviour of the two edges. Furthermore, the localisation and redistribution of known mechano-sensors (CAV-1 and Class I-Myosins) during linear motility confirmed differences in tension profiles. We found that the membrane tension profiles of the two cell edges are not synchronised during Glioblastoma linear migration, reflecting their uncoupled dynamics. Interestingly, cholesterol content inversely correlated with the motile behaviour of Glioblastoma cell lines; fast-motile cell lines showed higher cholesterol content than non-motile ones. Moreover, cholesterol clusters distributed differently between the cell front and rear of migrating Glioblastoma cells, matching their cell edges' tension profiles. This result highlighted a putative role of cholesterol clusters in creating tension differentials as insulators of membrane tension propagation between different cell regions. These findings pave the way to reconsidering years of literature around actin-based mechanical processes at the cell leading edge, underestimating cholesterol-rich membrane domains' contribution to tension propagation to the cell trailing edge. This PhD thesis has added evidence on membrane tension's role in the mechanical control of cell motility and cholesterol participation in Glioblastoma invasion.

Publisher

University of Galway

Rights

Attribution-NonCommercial-NoDerivatives 4.0 International

cbn

Collections

University of Galway Theses (PhD Theses)