Biophysical Modelling

BII - Biophysical Modelling Group Photo

Research

Our group uses a combination of biophysical and bioimaging techniques to study cell migration. Cell migration is a critical process in every living organism. For example, when wounds heal, skin cells migrate to close the wound. During cancer metastasis, cancer cells migrate and spread to a distant part of the body to initiate the formation of a new tumor. We develop novel assays to measure the biophysical properties of cell migration in three-dimensional microenvironments, and elucidate the signaling and gene regulatory networks that govern these properties, especially the role of the cytoskeleton. From these studies, we hope to identify novel therapeutic targets to perturb cell migration, which can eventually be translated into drugs to promote wound healing or stop cancer cell invasion.

Recently, we have looked at the migration of neutrophils. Neutrophils are white blood cells that play an important role in the host defence against pathogenic microbial agents. Under resting conditions, approximately 98% of the mature neutrophils in the body are stored within the bone marrow, with approximately 2% of the total mature neutrophils distributed in the blood stream and tissues. Upon exposure to inflammatory stimuli, neutrophils are rapidly mobilized from the bone marrow into the circulation, and are recruited to the site of inflammation where they carry out their functions and promote tissue remodelling. The distribution of neutrophils between bone marrow and blood compartment is mediated by egression and retention signals provided by chemokines CXCL2 and CXCL12, respectively. In bone marrow, CXCL12 constitutively expressed by stroma cells promotes neutrophil accumulation in the bone marrow through interaction with CXCR4. Moreover, the CXCR4-CXCL12 signalling axis also activates several signalling processes such as cell migration and proliferation.

Defects in neutrophil mobilization from the bone marrow into circulation leads to recurrent infections, as evinced by patients with the Warts, Hypogammaglobulinemia, Infections, and Myelokathexis (WHIM) syndrome. WHIM syndrome is an inherited primary immunodeficiency disease that subject the affected individual to recurrent bacterial infections due to low counts of most leukocytes, including neutrophils, circulating in the blood. WHIM patients expressing truncated CXCR4 display active receptor signalling that decays over a longer time as compared to WT leukocytes upon ligand stimulation. It has been shown that CXCL12-activated WHIM leukocytes show sustained activation of downstream signalling pathways such as pERK with greater signalling amplitude. Mutant leukocytes also exhibit higher sensitivity to CXCL12 as a larger percentage of cells migrated towards CXCL12 in transwell assays compared to WT cells.

BII - Biophysical Modelling Figure 1

Figure 1 : Morphological characterization of WT and WHIM neutrophils. (a) DIC images of WT and WHIM neutrophils with no protrusions, filopodia-type protrusions (arrows), and lamellipodia-type protrusions (arrowheads) before and after CXCL12 stimulation. Scale bars denote 5 µm. (b-c) Percentage of time that each cell produce no protrusion, filopodia-type or lamellipodia-type protrusion as observed over 15 min period before CXCL12 stimulation and 30 min period post-CXCL12 stimulation for (b) WT (n=25) and (c) WHIM neutrophils (n=23). Error bars denote standard error of the mean. *,** and **** represents p <0.05, p <0.01 and p <0.001 respectively. (d) Mean speed of WT (n=25) and WHIM (n=23) neutrophils exhibiting filopodia- or lamellipodia-type protrusion after CXCL12 stimulation. Error bars denote standard error of the mean. * represents p <0.05.

To understand neutrophil migration in WHIM patients, we have characterized the mechanochemical properties of wild type (WT) and WHIM neutrophils (Figure 1) and show that neutrophils isolated from WHIM mouse model are primed for cell migration. These mutant cells exhibit higher traction stresses, faster migration speed, and form more lamellipodia on stimulation with CXCL12 as compared to WT cells. Filopodia and lamellipodia are typically formed as a cell migrate. Specifically, filopodia protrusions are associated with extracellular matrix sensing to identify targets for adhesion which may mature into larger adhesion structures upon lamellipodia advancement. Lamellipodia, on the other hand, are associated with efficient cell migration. Hence, increased frequency of lamellipodia-type protrusions in WHIM neutrophils may result in increase in cell migration speed. In agreement, we found that cells forming the lamellipodia-type of protrusions tend to migrate at a faster speed than cells forming the filopodia-type protrusions. WHIM neutrophils are also characterized by larger initial increase in cell migration speed upon ligand stimulation, which decreases more slowly over time as compared to WT neutrophils, suggesting both increased CXCR4 sensitivity to CXCL12 and a longer CXCR4 internalization adaptation time.

BII - Biophysical Modelling Figure 2

Figure 2 : Mechanochemical model of neutrophil migration. (a) Schematic diagram of the signalling pathway involving CXCR4, CXCL12-activated CXCR4 (CXCR4*) and CXCR4* bound to β-arrestin (CXCR4*-β). By solving the kinetic mass action equations for the reactants in the signalling pathway, the maximum [CXCR4*] value achieved, termed CXCR4 sensitivity α, and the time scale of the decay in [CXCR4*], termed CXCR4 internalization adaptation time tadapt, can be written in terms of reaction rate parameters k1 and k2. (b) Schematic diagram of the negative integral feedback control system to obtain direction of the perceived gradient g based on values of the α and tadapt obtained from the reaction rate parameters k1 and k2. (c) Graph showing how tadapt change as k2 is varied. (d) Graph showing how α change as k2 is varied. (e) Cell speed vs. time post-CXCL12 stimulation for WT (empty circles) and WHIM (solid squares) neutrophils. Dotted lines show the corresponding two exponential model fit to the experimental cell speed measurements. n = 25 and n = 23 for WT and WHIM respectively. Error bars denote standard error of the mean. (f) Percentage of the cells in the bone marrow after 500 min in the computational model at various values of k1 and k2. (g) Trajectories, over 7 min, of a cell with a constant α=0.983, but different values of tadapt (Short: tadapt = 1.67 min; Intermediate: tadapt = 12.9 min; Long: tadapt = 167 min). (h) Trajectories, over 15 min, of a cell with a constant tadapt =167 min, but different values of α (Low: α = 0.465; Intermediate: α = 0.794 High: α = 0.983). Concentration of CXCL12 is represented by the gray intensity, with increasing gray levels denoting increasing levels of [CXCL12].

To understand how increased CXCR4 internalization adaptation time and CXCR4 sensitivity to CXCL12 enhances WHIM neutrophil accumulation in the bone marrow, we have proposed a mechanochemical model of the CXCR4-CXCL12 interaction, incorporating CXCR4 activation by CXCL12 and CXCR4 receptor internalization through β-arrestin-mediated endocytosis (Figure 2). The model explained how reducing the receptor internalization rate, as in the case in WHIM neutrophils, can result in longer CXCR4 internalization adaptation time and increased CXCR4 sensitivity to CXCL12. Through the model, we also explained that both longer CXCR4 internalization adaptation times and increased CXCR4 sensitivity collectively contribute to the increased accumulation of WHIM neutrophils in the bone marrow. We have also proposed, through traction stress measurements, that WHIM neutrophils are more adherent to the substratum which may contribute to the increased accumulation of WHIM neutrophils in the bone marrow. Lastly, differences in the type of cell protrusions produced by WT and WHIM neutrophils may suggest differences in the signalling pathways involved in the activation of the ras-related superfamily of small GTPase (Rac and Cdc42). Although more work is necessary to elucidate the differences in cell-substrate adhesions and small GTPase activation due to CXCR4-CXCL12 signalling in WT and WHIM neutrophils, our work has offered a mechanistic framework to understand WT and WHIM neutrophil migration and accumulation in the bone marrow.

Members

 Senior Principal Investigator  CHIAM Keng Hwee   |    [View Bio]   
 Research Officer GOH Jie Hui Corinna
 Research Officer ZHOU Tianxun
 PhD Student JUN Myeongjun 

Selected Publications