138 research outputs found
Developing a long-term microfluidic system to study the mesoscopic dynamics of Piezo1 activity
No full textThe cellular microenvironment (or cell niche) is a complex and specialised environment, consisting of physical, chemical, and biological factors that collectively influence the cells residing within. Interactions within this dynamic microenvironment play a critical role in regulating and maintaining cellular activities and function. Within the cellular microenvironment, resident cells constantly experience various mechanical force stimuli, which previous in vitro studies cannot fully recapitulate. Hence, the inclusion of mechanical forces (for example fluid shear stress) in the cell culturing system would yield a more physiologically relevant investigation of cellular behaviour.
Cells commonly express mechanosensitive proteins and receptors to sense and transduce external mechanical stimuli to respond appropriately. A widely expressed mechanosensitive ion channel, Piezo1, has been shown to play an important role in cellular mechanotransduction. Our lab previously generated a genetically-encoded Piezo1 sensor (GenEPi), which allows precise detection of Piezo1-specific activities. To investigate the activities of Piezo1 in near-physiological conditions, I developed a microfluidic-based system to study the dynamics of Piezo1 in mammalian cells using our GenEPi sensor.
Using my developed system, I discovered that Piezo1 clusters exhibit more directed diffusive motion after fluid shear stress. By studying the dynamics of the protein, I revealed differential Piezo1 responses across fluid shear stress magnitudes. I also reported a mechanical threshold to HEK-Piezo1 activation during constant fluid shear stress. Through the GenEPi dynamic responses, I observed that cellular Piezo1 activations are synchronised to mechanical shear triggers but become increasingly asynchronous under sustained shear stress. Crucially, the characteristics of Piezo1’s dynamic within cells reported here will significantly advance our understanding of the interplay between mechanical forces and cellular behaviour within the cellular microenvironment. This system can also be a model for others to adopt in investigating the dynamics of other mechanosensitive proteins expressed in the cells.Open Acces
High-Resolution Functional Imaging of Native Proteins using Force Distance Curve Based Atomic Force Microscopy
No full textRole of endocytic trafficking during Dpp gradient formation
No full textMorphogens are secreted signalling molecules that are expressed in restricted groups of cells within the developing tissue. From there, they are secreted and travel throughout the target field and form concentration gradients. These concentration profiles endow receiving cells with positional information. A number of experiments in Drosophila demonstrated that the morphogen Decapentaplegic (Dpp) forms activity gradients by inducing the expression of several target genes above distinct concentration thresholds at different distances from the source. This way, Dpp contributes to developmental fates in the target field such as the Drosophila wing disc. Although the tissue distribution as well as the actual shape and size of the Dpp morphogen concentration gradient has been visualized, the cell biological mechanisms through which the morphogen forms and maintains a gradient are still a subject of debate. Two hypotheses as to the dominant mechanism of movement have been proposed that can account for Dpp spreading throughout the Drosophila wing imaginal target tissue: extracellular diffusion and planar transcytosis, i. e. endocytosis and resecretion of the ligand that is thereby transported through the cells. Here, I present data indicating that implications of a theoreticalanalysis of Dpp spreading, where Dpp transport through the target tissue is solely based on extracellular diffusion taking into account receptor binding and subsequent internalization, are inconsistent with experimental results. By performing Fluorescence Recovery After Photobleaching (FRAP) experiments, I demonstrate a key role of Dynamin-mediated endocytosis for Dpp gradient formation. In addition, I show that most of GFP-Dpp traffics through endocytic compartments at the receiving epithelial cells, probably recycled through apical recycling endosomes (ARE). Finally, a Dpp recycling assay based on subcellular photouncage of ligand is presented to address specifically the Dpp recycling event at the receiving cells
PhOTO-Bow: An Integrative Transgenic Strategy for Single-Cell Tracking with Clonal Analysis
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Primed Track: Reliable Volumetric Single-cell Tracking and Lineage Tracing of Living Specimen with Dual-labeling Approaches
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