Cells and force transduction

This thesis studies mechanism involved in propagating force generated at cadherin complexes. The first part of this thesis demonstrates that mechanotransduction at classical cadherin complexes is not only ligand-dependent but also dependent on the respective receptor tyrosine kinase (RTK) binding partner of cadherin. This involvement of RTKs at cadherin complexes is important in propagating force transduction globally, implying that force transduction at cadherin complexes is not restricted to cell-cell junctions but is also propagated globally via the mediation of its respective RTK binding partner. These results suggest that homophilic ligation in trans- and cadherin association with cognate receptor tyrosine kinase in cis comprises a combinatorial, mechano-chemical switch. That is, specific combinations of cadherin, ligand, and RTK is required for force-activated RTK-dependent signaling, activation of cell contractility, and cytoskeletal remodeling at perturbed cadherin adhesions. These findings confirm that cadherins form both homophilic and heterophilic bonds, but homophilic cadherin ligation selectively triggers cadherin-associated RTK signals that mechanically reinforce homophilic, but not heterophilic cadherin adhesions, thereby stabilizing homophilic adhesions and amplifying binding differences. This study demonstrates that this mechano-chemical switch is not governed by cadherin adhesion differences, but requires a specific combination of cadherin ligand in trans- and RTK expression in cis to actuate force transduction signaling on rigid surfaces to propagate force transduction at a global level. For the second part of this study used novel, force-limited nanoscale tension gauges to investigate how force and substrate stiffness guide cellular decision-making during initial cell attachment and spreading on deformable substrates. The well-established dependence of cell traction and spreading on substrate stiffness has been attributed to levels of force exerted on molecular components in focal contacts. The molecular tension gauges used in this study enabled direct estimates of the threshold, pico Newton forces that instructed decision-making at different stages of cell attachment, spreading, and adhesion maturation. These results further confirm that the force thresholds controlling adhesion and spreading transitions depend on substrate stiffness. Reported findings agree semi-quantitatively with a proposed model that attributes rigidity-dependent differences in cell spreading to stiffness-dependent rates of competing biochemical processe

Cellular decision making at the nanoscale

The well-established dependence of cell traction forces on the compliance of supporting matrices has been attributed to levels of force exerted on components in focal contacts. Here, use of novel, force-limited nanoscale tension gauges revealed that both force and substrate deformations govern cell decision-making during initial attachment to compliant substrates. We propose a mechanical model consistent with observed behavior. Upon formation of stable cell contacts, bond tension and tether rupture govern cell attachment, spreading, and focal adhesion maturation at force levels on individual receptors predicted by prior studies

Constructing Modular and Universal Single Molecule Tension Sensor Using Protein G to Study Mechano-sensitive Receptors

Recently a variety of molecular force sensors have been developed to study cellular forces acting through single mechano-sensitive receptors. A common strategy adopted is to attach ligand molecules on a surface through engineered molecular tethers which report cell-exerted tension on receptor-ligand bonds. This approach generally requires chemical conjugation of the ligand to the force reporting tether which can be time-consuming and labor-intensive. Moreover, ligand-tether conjugation can severely reduce the activity of protein ligands. To address this problem, we developed a Protein G (ProG)-based force sensor in which force-reporting tethers are conjugated to ProG instead of ligands. A recombinant ligand fused with IgG-Fc is conveniently assembled with the force sensor through ProG:Fc binding, therefore avoiding ligand conjugation and purification processes. Using this approach, we determined that molecular tension on E-cadherin is lower than dsDNA unzipping force (nominal value: 12 pN) during initial cadherin-mediated cell adhesion, followed by an escalation to forces higher than 43 pN (nominal value). This approach is highly modular and potentially universal as we demonstrate using two additional receptor-ligand interactions, P-selectin & PSGL-1 and Notch & DLL1

Differential Effects of Influenza Virus NA, HA Head, and HA Stalk Antibodies on Peripheral Blood Leukocyte Gene Expression during Human Infection.

In this study, we examined the relationships between anti-influenza virus serum antibody titers, clinical disease, and peripheral blood leukocyte (PBL) global gene expression during presymptomatic, acute, and convalescent illness in 83 participants infected with 2009 pandemic H1N1 virus in a human influenza challenge model. Using traditional statistical and logistic regression modeling approaches, profiles of differentially expressed genes that correlated with active viral shedding, predicted length of viral shedding, and predicted illness severity were identified. These analyses further demonstrated that challenge participants fell into three peripheral blood leukocyte gene expression phenotypes that significantly correlated with different clinical outcomes and prechallenge serum titers of antibodies specific for the viral neuraminidase, hemagglutinin head, and hemagglutinin stalk. Higher prechallenge serum antibody titers were inversely correlated with leukocyte responsiveness in participants with active disease and could mask expression of peripheral blood markers of clinical disease in some participants, including viral shedding and symptom severity. Consequently, preexisting anti-influenza antibodies may modulate PBL gene expression, and this must be taken into consideration in the development and interpretation of peripheral blood diagnostic and prognostic assays of influenza infection

Landscape of coordinated immune responses to H1N1 challenge in humans

Influenza is a significant cause of morbidity and mortality worldwide. Here we show changes in the abundance and activation states of more than 50 immune cell subsets in 35 individuals over 11 time points during human A/California/2009 (H1N1) virus challenge monitored using mass cytometry along with other clinical assessments. Peak change in monocyte, B cell, and T cell subset frequencies coincided with peak virus shedding, followed by marked activation of T and NI< cells. Results led to the identification of C038 as a critical regulator of plasmacytoid dendritic cell function in response to influenza virus. Machine learning using study-derived clinical parameters and single-cell data effectively classified and predicted susceptibility to infection. The coordinated immune cell dynamics defined in this study provide a framework for identifying novel correlates of protection in the evaluation of future influenza therapeutics

Cells and force transduction

This thesis studies mechanism involved in propagating force generated at cadherin complexes. The first part of this thesis demonstrates that mechanotransduction at classical cadherin complexes is not only ligand-dependent but also dependent on the respective receptor tyrosine kinase (RTK) binding partner of cadherin. This involvement of RTKs at cadherin complexes is important in propagating force transduction globally, implying that force transduction at cadherin complexes is not restricted to cell-cell junctions but is also propagated globally via the mediation of its respective RTK binding partner. These results suggest that homophilic ligation in trans- and cadherin association with cognate receptor tyrosine kinase in cis comprises a combinatorial, mechano-chemical switch. That is, specific combinations of cadherin, ligand, and RTK is required for force-activated RTK-dependent signaling, activation of cell contractility, and cytoskeletal remodeling at perturbed cadherin adhesions. These findings confirm that cadherins form both homophilic and heterophilic bonds, but homophilic cadherin ligation selectively triggers cadherin-associated RTK signals that mechanically reinforce homophilic, but not heterophilic cadherin adhesions, thereby stabilizing homophilic adhesions and amplifying binding differences. This study demonstrates that this mechano-chemical switch is not governed by cadherin adhesion differences, but requires a specific combination of cadherin ligand in trans- and RTK expression in cis to actuate force transduction signaling on rigid surfaces to propagate force transduction at a global level. For the second part of this study used novel, force-limited nanoscale tension gauges to investigate how force and substrate stiffness guide cellular decision-making during initial cell attachment and spreading on deformable substrates. The well-established dependence of cell traction and spreading on substrate stiffness has been attributed to levels of force exerted on molecular components in focal contacts. The molecular tension gauges used in this study enabled direct estimates of the threshold, pico Newton forces that instructed decision-making at different stages of cell attachment, spreading, and adhesion maturation. These results further confirm that the force thresholds controlling adhesion and spreading transitions depend on substrate stiffness. Reported findings agree semi-quantitatively with a proposed model that attributes rigidity-dependent differences in cell spreading to stiffness-dependent rates of competing biochemical processesLimitedAuthor requested closed access (OA after 2yrs) in Vireo ETD syste

A novel isoform of MAP4 organises the paraxial microtubule array required for muscle cell differentiation

The microtubule cytoskeleton is critical for muscle cell differentiation and undergoes reorganisation into an array of paraxial microtubules, which serves as template for contractile sarcomere formation. Here, we identify a previously uncharacterised isoform of microtubule-associated protein MAP4, oMAP4, as a microtubule organising factor that is crucial for myogenesis. We show that oMAP4 is expressed upon muscle cell differentiation and is the only MAP4 isoform essential for normal progression of the myogenic differentiation programme. Depletion of oMAP4 impairs cell elongation and cell-cell fusion. Most notably, oMAP4 is required for paraxial microtubule organisation in muscle cells and prevents dynein- and kinesin-driven microtubule-microtubule sliding. Purified oMAP4 aligns dynamic microtubules into antiparallel bundles that withstand motor forces in vitro. We propose a model in which the cooperation of dynein-mediated microtubule transport and oMAP4-mediated zippering of microtubules drives formation of a paraxial microtubule array that provides critical support for the polarisation and elongation of myotubes

Differential Effects of Influenza Virus NA, HA Head, and HA Stalk Antibodies on Peripheral Blood Leukocyte Gene Expression during Human Infection

Influenza A viruses are significant human pathogens that caused 83,000 deaths in the United States during 2017 to 2018, and there is need to understand the molecular correlates of illness and to identify prognostic markers of viral infection, symptom severity, and disease course. Preexisting antibodies against viral neuraminidase (NA) and hemagglutinin (HA) proteins play a critical role in lessening disease severity. We performed global gene expression profiling of peripheral blood leukocytes collected during acute and convalescent phases from a large cohort of people infected with A/H1N1pdm virus. Using statistical and machine-learning approaches, populations of genes were identified early in infection that correlated with active viral shedding, predicted length of shedding, or disease severity. Finally, these gene expression responses were differentially affected by increased levels of preexisting influenza antibodies, which could mask detection of these markers of contagiousness and disease severity in people with active clinical disease.In this study, we examined the relationships between anti-influenza virus serum antibody titers, clinical disease, and peripheral blood leukocyte (PBL) global gene expression during presymptomatic, acute, and convalescent illness in 83 participants infected with 2009 pandemic H1N1 virus in a human influenza challenge model. Using traditional statistical and logistic regression modeling approaches, profiles of differentially expressed genes that correlated with active viral shedding, predicted length of viral shedding, and predicted illness severity were identified. These analyses further demonstrated that challenge participants fell into three peripheral blood leukocyte gene expression phenotypes that significantly correlated with different clinical outcomes and prechallenge serum titers of antibodies specific for the viral neuraminidase, hemagglutinin head, and hemagglutinin stalk. Higher prechallenge serum antibody titers were inversely correlated with leukocyte responsiveness in participants with active disease and could mask expression of peripheral blood markers of clinical disease in some participants, including viral shedding and symptom severity. Consequently, preexisting anti-influenza antibodies may modulate PBL gene expression, and this must be taken into consideration in the development and interpretation of peripheral blood diagnostic and prognostic assays of influenza infection