Research
Uncovering fundamental mechanisms underlying "dynamic" mechanotransduction
Cells in our body are exposed to various mechanical forces from their neighbors and environment: for example, endothelial cells lining in the blood vessel are exposed to shear stress and pulsatile pressure from the blood flow. At the basal surface of the cell, however, cells are interfacing with something called extracellular matrix (ECM), which supports the cell not only chemically but also mechanically. In recent 20 years, it has been revealed that the rigidity of the extracellular matrix can greatly influence physiology and pathology of cells and tissues, including differentiation, survival, proliferation, altered drug response, and tumor progression. For example, in the case of tumor, the increase in the tissue stiffness without any changes in genetic information and chemical environment, can cause tumor progression. There is also an evidence showing that cancer-targeting drug does not work when cancer cells are highly contractile in very tensed environment.
Fig. 1. Dynamic and heterogeneous adhesion assembly and disassembly in the protrusion of Chinese Hamster Ovary epithelial cell, classified by Machine Learning
Mechanical sensing of the ECM by cells occurs through cell-matrix adhesions that link the ECM to the cytoskeleton. As a mechanical anchor, cell-matrix adhesions transmit mechanical forces from the actin cytoskeleton to the ECM. At the same time, they transduce the force they “felt” into biochemical signals, cellular response to which lead to changes in a wide variety of cell functions including structural reinforcement. While structure, function, and signaling by adhesions have been characterized at the level of large, mature focal adhesions, however, it has been elusive whether mechanosensing can occur before they reach their full maturation: i.e. in the state of nascent adhesions. Indeed these adhesions are very dynamic: they constantly form, turn over and mature (Fig.1).
Fig.2. (Left) vinculin of a CHO cell (green) and fluorescent beads on the gel (green) overlaid with traction vectors. (Right) Color coded of traction magnitude (blue to red, low to high) overlaid with traction vectors. Image credit: Alexia Bachir.
To study this dynamic mechanotransduction, 1) a tool to resolve small force from tiny adhesions and 2) a framework that allows linking mechanical force to highly heterogeneous molecular events are required. We address these issues experimentally and computationally. First we develop traction force microscopy (TFM) algorithm that is very sensitive to small forces from nascent adhesions as well as big forces from focal adhesions (Fig. 2.) while insensitive to noise in non-cell area, by introducing sparsity regularization into the inverse problem solution.
Second, we address the heterogeneity in the population of nascent adhesions with single particle tracking, machine learning and general image analysis of microscopic images (Fig. 3.). The measurements from these tools suggest evidence of independent force-transmission and mechano-sensitivity of a significant subset of nascent adhesions and highlight differential recruitment of early adhesion molecules (e.g. talin, vinculin, and paxillin).
We are expanding the framework of the TFM and adhesion-tracking-and-classification to 1) dynamic ECM rigidity sensing, 2) shear flow mechanotransduction and 3) cancer cell progression and metastasis.
Fig.3. Machine Learning of nascent and focal adhesions.
ECM Stiffness Sensing Mechanism
For stiffness sensing, we focus on the nascent adhesions and their role in ECM stiffness sensing and how sensed force can be transduced to biochemical signaling. The findings from the first funding period are well-described in our recent publication (Mittal et al., 2024) where we report stiffness-dependent differential traction is independent of myosin contractility and is regulated by actin polymerization and its contribution to F-actin network rigidity. We will keep pursuing finding general stiffness-dependent mechanotransduction pathways using additional quantification of signaling events in the cells.
Fluid Shear Stress Sensing Mechanism
Inspired by my previous work (Ting et al., 2012) and the classic hypothesis by Peter Davies (Davies, 1995), we started to step into the endothelial mechanotransduction field by measuring traction during dynamically varying fluid shear stress. My doctoral student Mohanish recently published an article about the role of temporary low fluid shear stress in traction modulation and cell alignment (Chandurkar et al., 2024). We aim to investigate the upstream regulators and downstream effectors of endothelial contractility. Paul Evans (QMUL) is an consultant in this project.
Mechanobiology involved in Ehlers-Danlos Syndrome
The classical type Ehlers-Danlos Syndrome (cEDS) is characterized by stretchy skin and hyper-mobile joints. We hypothesize that impaired wound healing in cEDS might be attributed to changed local mechanical properties of the ECM caused by insufficient production of collagen type V (Royer and Han, 2022). We extend this idea to improve wound healing with external collagen type V addition with further collagen-stiffening reagents.