Allen Liu

Mechanical and biochemical regulation of clathrin-mediated endocytosis

Clathrin-mediated endoctyosis (CME) maintains cellular and organismal homeostasis by mediating the uptake of nutrients and controlling the expression and activity of signaling receptors. The fundamental functional unit of CME is a clathrin-coated pit (CCP) and the dynamic behavior of CCPs has been observed to be highly heterogeneous. We are interested in how mechanical and chemical stimuli affect the behaviors of CCP internalization. Recently, we have shown that CCP dynamics are in part regulated by cell tension, though the interplay between biochemical and mechanical regulation is likely to be highly complex. A major motivation for studying endocytosis is our general hypothesis that endocytic regulation of plasma membrane receptors regulates various aspects of directed cell migration. In this regards, we seek to define the biochemical regulation of activated chemokine receptor CXCR4 internalization, with the long-term goal of understanding the dynamics of membrane trafficking and signaling during cell motility. The combination of total internal reflection fluorescence microscopy (TIR-FM) and computational analysis under controlled mechanical and biochemical perturbations represents a powerful approach to understand what regulates the formation of individual CCPs. We are currently using breast cancer cells as a model system to explore the link between endocytosis and cell migration. We are also conducting high throughput imaging and analysis to understand how endocytic phenotypes influence cell migration phenotypes as well as employing a proteomics approach to investigate how compositional changes in CCPs are regulated under different mechanical and biochemical conditions.

Building artificial platelets

Bottom-up synthetic biology is the systematic construction of functional biological systems with biomolecules. The critical accumulation of our knowledge about individual biomolecules can enable us to integrate them into a system in a meaningful way to create cellular devices. Artificial cells, like any other engineered systems, are merely a device that senses input information (e.g. forces, molecules, light…. etc.) and provide a specific output (e.g. shape change, enzymatic activities, secretion…etc.). We have identified platelets as a tractable biological system to emulate through modular design. Our design strategy necessitates an understanding of the functionalities of natural platelets so that our artificial platelets can confer the essential functions of natural platelets. Central to our idea of artificial platelets is a coupling of forces and enzymatic activities, and we are currently using mechanosensitive channel of large conductance (MscL) as a model mechanosensitive protein for this application. The platelets will be made as lipid vesicles that have defined lipid and protein compositions using droplet microfluidics, and we have also developed a mammalian cell free expression system for producing proteins of interests. The power of combining vesicle encapsulation and cell-free expression will bring about a new paradigm of building minimal cells and reconstitution of biological functions.

Cellular Mechanotransduction

A mechanism underpinning the activation of our artificial platelets described previously depends on the functional reconstitution of a bacterial mechanosensitive channel MscL. MscL responds to membrane tension and acts as a non-selective channel for the transit of small molecules. We have investigated the functional reconstitution of MscL expressed in mammalian cells and found that forces applied to the focal adhesion-cytoskeleton linkage could induce the activation of MscL. Our work has inspired a new strategy for delivery of cell-impermeant small molecules and could have broad application in drug delivery and introducing mechanosensitive functions to cells. We are now repurposing MscL in breast cancer cells to test a 3D cell migration model. We are also testing MscL activation under different mechanical perturbations and collaborating with theorists to understand MscL activation under flow.

Microengineering Tools for Cell Mechanics Applications

We have been developing bioengineering tools to facilitate our research in mechanobiology of membrane and along this path have spurred a number of collaborations in applying microfluidic tools for cell mechanics applications. Mechanical properties of individual cells are altered in some diseases. While disease diagnosis still depends heavily on known biomarkers, there is more appreciation in recent years that cell stiffness could be used as a label-free biomarker. Moreover, it is well recognized now that physical stimuli regulate various cellular functions, including cell migration, cell division, and differentiation. To overcome limitations in conventional micropipette aspiration technique, we have developed a microfluidic micropipette aspiration device capable of parallel measurements where cell deformation is controlled only by the volumetric flow rate. We have applied this novel device for cell mechanical property measurement of breast cancer cells as well as determining the activation threshold of MscL expressing cells. The use microfluidic device for single cell analysis has transformative potential for clinical diagnosis. In addition, we have also devised a cell stretcher for in situ cell stretching while combining live cell imaging. We are currently developing new microfluidic tools for examining different aspects/modes of force transduction in MscL expressing cells.

Research Areas(s)

• Systems biology
• Synthetic biology
• Cytoskeleton dynamics
• Membrane organization
• Cellular biophysics

Award(s)

• NIH Director’s New Innovator Award (2012)
• Leukemia and Lymphoma Society Fellowship (2009)