tension-gauge-tether-probes-for-quantifying-growth-factor-mediated

Tension Gauge Tether Probes for Quantifying Growth Factor Mediated Integrin Mechanics and Adhesion

Multicellular organisms rely on interactions between membrane receptors and cognate ligands in the surrounding extracellular matrix (ECM) to orchestrate ...

University of Alabama at Birmingham

Communication among neurons is mediated through synaptic connections between axons and dendrites, and most excitatory synapses occur on actin-rich protrusions along dendrites called dendritic spines. Dendritic spines are structurally dynamic, and synapse strength is closely correlated with spine morphology. Abnormalities in the size, shape, and number of dendritic spines are prevalent in neurologic diseases, including autism spectrum disorders, schizophrenia, and Alzheimer disease. However, therapeutic targets that influence spine morphology are lacking. Rho-associated coiled-coil containing protein kinases (ROCK) 1 and ROCK2 are potent regulators of the actin cytoskeleton and highly promising drug targets for central nervous system disorders. In this report, we addressed how pharmacologic inhibition of ROCK1 and ROCK2 affects dendritic spine morphology. Hippocampal neurons were transfected with plasmids expressing fluorescently labeled Lifeact, a small actin binding peptide, and then incubated with or without Y-27632, an established pan-ROCK small molecule inhibitor. Using an automated 3D spine morphometry analysis method, we showed that inhibition of ROCK1 and ROCK2 significantly increased the mean protrusion density and significantly reduced the mean protrusion width. A trending increase in mean protrusion length was observed following Y-27632 treatment, and novel effects were observed among spine classes. Exposure to Y-27632 significantly increased the number of filopodia and thin spines, while the numbers of stubby and mushroom spines were similar to mock-treated samples. These findings support the hypothesis that pharmacologic inhibition of ROCK1 and ROCK2 may convey therapeutic benefit for neurologic disorders that feature dendritic spine loss or aberrant structural plasticity.

ROCK1 and ROCK2 inhibition alters dendritic spine morphology in hippocampal neurons.

Desmosomes are macromolecular cell-cell junctions that provide adhesive strength in epithelial tissue. Desmosome function is inseparably linked to structure, and it is hypothesized that the arrangement, or order, of desmosomal cadherins in the intercellular space is critical for adhesive strength. However, due to desmosome size, molecular complexity, and dynamics, the role that order plays in adhesion is challenging to study. Herein, we present an excitation resolved fluorescence polarization microscopy approach to measure the spatiotemporal dynamics of order and disorder of the desmosomal cadherin desmoglein 3 (Dsg3) in living cells. Simulations were used to establish order factor as a robust metric for quantifying the spatiotemporal dynamics of order and disorder. Order factor measurements in keratinocytes showed the Dsg3 extracellular domain is ordered at the individual desmosome, single cell, and cell population levels compared to a series of disordered controls. Desmosomal adhesion is Ca dependent, and reduction of extracellular Ca leads to a loss of adhesion measured by dispase fragmentation assay (λ = 15.1 min). Live cell imaging revealed Dsg3 order decreased more rapidly (λ = 5.5 min), indicating that cadherin order is not required for adhesion. Our results suggest that rapid disordering of cadherins can communicate a change in extracellular Ca concentration to the cell, leading to a downstream loss of adhesion. Fluorescence polarization is an effective bridge between protein structure and complex dynamics and the approach presented here is broadly applicable to studying order in macromolecular structures.

Desmoglein 3 Order and Dynamics in Desmosomes Determined by Fluorescence Polarization Microscopy.

Mechanical forces are integral to many biological processes; however, current techniques cannot map the magnitude and direction of piconewton molecular forces. Here, we describe molecular force microscopy, leveraging molecular tension probes and fluorescence polarization microscopy to measure the magnitude and 3D orientation of cellular forces. We mapped the orientation of integrin-based traction forces in mouse fibroblasts and human platelets, revealing alignment between the organization of force-bearing structures and their force orientations.

Mapping the 3D orientation of piconewton integrin traction forces.

Cell junctions are critical for cell adhesion and communication in epithelial tissues. It is evident that the cellular distribution, size, and architecture of cell junctions play a vital role in regulating function. These details of junction architecture have been challenging to elucidate in part due to the complexity and size of cell junctions. A major challenge in understanding these features is attaining high resolution spatial information with molecular specificity. Fluorescence microscopy allows localization of specific proteins to junctions, but with a resolution on the same scale as junction size, rendering internal protein organization unobtainable. Super-resolution microscopy provides a bridge between fluorescence microscopy and nanoscale approaches, utilizing fluorescent tags to reveal protein organization below the resolution limit. Here we provide a brief introduction to super-resolution microscopy and discuss novel findings into the organization, structure and function of epithelial cell junctions.

Bridging the gap: Super-resolution microscopy of epithelial cell junctions.

In systemic lupus erythematosus (SLE), type I IFNs promote induction of type I IFN-stimulated genes (ISG) and can drive B cells to produce autoantibodies. Little is known about the expression of distinct type I IFNs in lupus, particularly high-affinity IFN-β. Single-cell analyses of transitional B cells isolated from SLE patients revealed distinct B cell subpopulations, including type I IFN producers, IFN responders, and mixed IFN producer/responder clusters. Anti-Ig plus TLR3 stimulation of SLE B cells induced release of bioactive type I IFNs that could stimulate HEK-Blue cells. Increased levels of IFN-β were detected in circulating B cells from SLE patients compared with controls and were significantly higher in African American patients with renal disease and in patients with autoantibodies. Together, the results identify type I IFN-producing and -responding subpopulations within the SLE B cell compartment and suggest that some patients may benefit from specific targeting of IFN-β.

Cutting Edge: Intracellular IFN-β and Distinct Type I IFN Expression Patterns in Circulating Systemic Lupus Erythematosus B Cells.

Desmogleins (Dsgs) are cadherin family adhesion molecules essential for epidermal integrity. Previous studies have shown that desmogleins associate with lipid rafts, but the significance of this association was not clear. Here, we report that the desmoglein transmembrane domain (TMD) is the primary determinant of raft association. Further, we identify a novel mutation in the DSG1 TMD (G562R) that causes severe dermatitis, multiple allergies, and metabolic wasting syndrome. Molecular modeling predicts that this G-to-R mutation shortens the DSG1 TMD, and experiments directly demonstrate that this mutation compromises both lipid raft association and desmosome incorporation. Finally, cryo-electron tomography indicates that the lipid bilayer within the desmosome is ∼10% thicker than adjacent regions of the plasma membrane. These findings suggest that differences in bilayer thickness influence the organization of adhesion molecules within the epithelial plasma membrane, with cadherin TMDs recruited to the desmosome via the establishment of a specialized mesoscale lipid raft-like membrane domain.

The desmosome is a mesoscale lipid raft-like membrane domain.

Alzheimer's disease (AD) therapies predominantly focus on β-amyloid (Aβ), but Aβ effects may be maximal before clinical symptoms appear. Downstream of Aβ, dendritic spine loss correlates most strongly with cognitive decline in AD. Rho-associated kinases (ROCK1 and ROCK2) regulate the actin cytoskeleton, and ROCK1 and ROCK2 protein abundances are increased in early AD. Here, we found that the increased abundance of ROCK1 in cultured primary rat hippocampal neurons reduced dendritic spine length through a myosin-based pathway, whereas the increased abundance of ROCK2 induced spine loss through the serine and threonine kinase LIMK1. Aβ oligomers can activate ROCKs. Here, using static imaging studies combined with multielectrode array analyses, we found that the ROCK2-LIMK1 pathway mediated Aβ-induced spine degeneration and neuronal hyperexcitability. Live-cell microscopy revealed that pharmacologic inhibition of LIMK1 rendered dendritic spines resilient to Aβ oligomers. Treatment of hAPP mice with a LIMK1 inhibitor rescued Aβ-induced hippocampal spine loss and morphologic aberrations. Our data suggest that therapeutically targeting LIMK1 may provide dendritic spine resilience to Aβ and therefore may benefit cognitively normal patients that are at high risk for developing dementia.

Pharmacologic inhibition of LIMK1 provides dendritic spine resilience against β-amyloid.

Podosomes are ubiquitous cellular structures important to diverse processes including cell invasion, migration, bone resorption, and immune surveillance. Structurally, podosomes consist of a protrusive actin core surrounded by adhesion proteins. Although podosome protrusion forces have been quantified, the magnitude, spatial distribution, and orientation of the opposing tensile forces remain poorly characterized. Here we use DNA nanotechnology to create probes that measure and manipulate podosome tensile forces with molecular piconewton (pN) resolution. Specifically, Molecular Tension-Fluorescence Lifetime Imaging Microscopy (MT-FLIM) produces maps of the cellular adhesive landscape, revealing ring-like tensile forces surrounding podosome cores. Photocleavable adhesion ligands, breakable DNA force probes, and pharmacological inhibition demonstrate local mechanical coupling between integrin tension and actin protrusion. Thus, podosomes use pN integrin forces to sense and respond to substrate mechanics. This work deepens our understanding of podosome mechanotransduction and contributes tools that are widely applicable for studying receptor mechanics at dynamic interfaces.

DNA mechanotechnology reveals that integrin receptors apply pN forces in podosomes on fluid substrates.

A fundamental challenge with fluorophore orientation measurement is degeneracy, which is the inability to distinguish between multiple unique fluorophore orientations. Techniques exist for the non-degenerate measurement of the orientations of single, static fluorophores. However, such techniques are unsuitable for densely labeled and/or dynamic samples common to biological research. Accordingly, a rapid, widefield microscopy technique that can measure orientation parameters for ensembles of fluorophores in a non-degenerate manner is desirable. We propose that exciting samples with polarized light and multiple incidence angles could enable such a technique. We use Monte Carlo simulations to validate this approach for specific axially symmetric ensembles of fluorophores and obtain optimal experimental parameters for its future implementation.

Variable incidence angle linear dichroism (VALiD): a technique for unique 3D orientation measurement of fluorescent ensembles.

Desmosomes are cell-cell junctions that provide mechanical integrity to epithelial and cardiac tissues. Desmosomes have two distinct adhesive states, calcium-dependent and hyperadhesive, which balance tissue plasticity and strength. A highly ordered array of cadherins in the adhesive interface is hypothesized to drive hyperadhesion, but how desmosome structure confers adhesive state is still elusive. We employed fluorescence polarization microscopy to show that cadherin order is not required for hyperadhesion induced by pharmacologic and genetic approaches. FRAP experiments in cells treated with the PKCα inhibitor Gö6976 revealed that cadherins, plakoglobin, and desmoplakin have significantly reduced exchange in and out of hyperadhesive desmosomes. To test whether this was a result of enhanced keratin association, we used the desmoplakin mutant S2849G, which conferred reduced protein exchange. We propose that inside-out regulation of protein exchange modulates adhesive function, whereby proteins are "locked in" to hyperadhesive desmosomes while protein exchange confers plasticity on calcium-dependent desmosomes, thereby providing rapid control of adhesion.

Protein exchange is reduced in calcium-independent epithelial junctions.

Mechanical forces, growth factors and the extracellular matrix all play crucial roles in cell adhesion. To understand how epidermal growth factor receptor (EGFR) impacts the mechanics of adhesion, we employed tension gauge tether (TGT) probes displaying the integrin ligand cRGDfK and quantified integrin tension. EGF exposure significantly increased spread area, cell circularity, integrated integrin tension, mechanical rupture density, radial organization and size of focal adhesions in Cos-7 cells on TGT surfaces. These findings suggest that EGFR regulates integrin tension and the spatial organization of focal adhesions. Additionally, we found that the mechanical tension threshold for outside-in integrin activation is tunable by EGFR. Parallel genetic and pharmacologic strategies demonstrated that these phenotypes are driven by ligand-dependent EGFR signaling. Our results establish a novel mechanism whereby EGFR regulates integrin activation and cell adhesion, providing control over cellular responses to the environment.This article has an associated First Person interview with the first author of the paper.

EGFR activation attenuates the mechanical threshold for integrin tension and focal adhesion formation.

Despite the vital role of mechanical forces in biology, it still remains a challenge to image cellular force with sub-100-nm resolution. Here, we present tension points accumulation for imaging in nanoscale topography (tPAINT), integrating molecular tension probes with the DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) technique to map piconewton mechanical events with ~25-nm resolution. To perform live-cell dynamic tension imaging, we engineered reversible probes with a cryptic docking site revealed only when the probe experiences forces exceeding a defined mechanical threshold (~7-21 pN). Additionally, we report a second type of irreversible tPAINT probe that exposes its cryptic docking site permanently and thus integrates force history over time, offering improved spatial resolution in exchange for temporal dynamics. We applied both types of tPAINT probes to map integrin receptor forces in live human platelets and mouse embryonic fibroblasts. Importantly, tPAINT revealed a link between platelet forces at the leading edge of cells and the dynamic actin-rich ring nucleated by the Arp2/3 complex.

Live-cell super-resolved PAINT imaging of piconewton cellular traction forces.

Desmosomes are cell-cell junctions responsible for mechanically integrating adjacent cells. Due to the small size of the junctions, their protein architecture cannot be elucidated using conventional fluorescence microscopy. Super-resolution microscopy techniques, including dSTORM, deliver higher-resolution images which can reveal the localization or arrangement of proteins within individual desmosomes. Herein we describe an imaging and analysis method to determine the nanoscale architecture of desmosomes using super-resolution dSTORM.

dSTORM Imaging and Analysis of Desmosome Architecture.

The nucleolus of a cell is a critical cellular compartment that is responsible for ribosome biogenesis and plays a central role in tumor progression. Fisetin, a nutraceutical, is a naturally occurring flavonol from the flavonoid group of polyphenols that has anti-cancer effects. Fisetin negatively impacts several signaling pathways that support tumor progression. However, effect of fisetin on the nucleolus and its functions were unknown. We observed that fisetin is able to physically enter the nucleolus. In the nucleolus, RNA polymerase I (RNA Pol I) mediates the biogenesis of ribosomal RNA. Thus, we investigated the impacts of fisetin on the nucleolus. We observed that breast tumor cells treated with fisetin show a 20-30% decreased nucleolar abundance per cell and a 30-60% downregulation of RNA Pol I transcription activity, as well as a 50-70% reduction in nascent rRNA synthesis, depending on the cell line. Our studies show that fisetin negatively influences MAPK/ERK pathway to impair RNA Pol I activity and rRNA biogenesis. Functionally, we demonstrate that fisetin acts synergistically (CI = 0.4) with RNA Pol I inhibitor, BMH-21 and shows a noteworthy negative impact (60% decrease) on lung colonization of breast cancer cells. Overall, our findings highlight the potential of ribosomal RNA (rRNA) biogenesis as a target for secondary prevention and possible treatment of metastatic disease.

Novel role of the dietary flavonoid fisetin in suppressing rRNA biogenesis.

Many cellular processes, including cell division, development, and cell migration require spatially and temporally coordinated forces transduced by cell-surface receptors. Nucleic acid-based molecular tension probes allow one to visualize the piconewton (pN) forces applied by these receptors. Building on this technology, we recently developed molecular force microscopy (MFM) which uses fluorescence polarization to map receptor force orientation with diffraction-limited resolution (~250 nm). Here, we show that structured illumination microscopy (SIM), a super-resolution technique, can be used to perform super-resolution MFM. Using SIM-MFM, we generate the highest resolution maps of both the magnitude and orientation of the pN traction forces applied by cells. We apply SIM-MFM to map platelet and fibroblast integrin forces, as well as T cell receptor forces. Using SIM-MFM, we show that platelet traction force alignment occurs on a longer timescale than adhesion. Importantly, SIM-MFM can be implemented on any standard SIM microscope without hardware modifications.

Turn-key mapping of cell receptor force orientation and magnitude using a commercial structured illumination microscope.

Author Correction: Turn-key mapping of cell receptor force orientation and magnitude using a commercial structured illumination microscope.

Desmosomes are macromolecular cell-cell junctions critical for maintaining adhesion and resisting mechanical stress in epithelial tissue. Desmosome assembly and the relationship between maturity and molecular architecture are not well understood. To address this, we employed a calcium switch assay to synchronize assembly followed by quantification of desmosome nanoscale organization using direct Stochastic Optical Reconstruction Microscopy (dSTORM). We found that the organization of the desmoplakin rod/C-terminal junction changed over the course of maturation, as indicated by a decrease in the plaque-to-plaque distance, while the plaque length increased. In contrast, the desmoplakin N-terminal domain and plakoglobin organization (plaque-to-plaque distance) were constant throughout maturation. This structural rearrangement of desmoplakin was concurrent with desmosome maturation measured by E-cadherin exclusion and increased adhesive strength. Using two-color dSTORM, we showed that while the number of individual E-cadherin containing junctions went down with the increasing time in high Ca, they maintained a wider desmoplakin rod/C-terminal plaque-to-plaque distance. This indicates that the maturation state of individual desmosomes can be identified by their architectural organization. We confirmed these architectural changes in another model of desmosome assembly, cell migration. Desmosomes in migrating cells, closest to the scratch where they are assembling, were shorter, E-cadherin enriched, and had wider desmoplakin rod/C-terminal plaque-to-plaque distances compared to desmosomes away from the wound edge. Key results were demonstrated in three cell lines representing simple, transitional, and stratified epithelia. Together, these data suggest that there is a set of architectural programs for desmosome maturation, and we hypothesize that desmoplakin architecture may be a contributing mechanism to regulating adhesive strength.

Article	Year
ROCK1 and ROCK2 inhibition alters dendritic spine morphology in hippocampal neurons. Cellular logistics\| PubMed ID: 27054047	2015
Desmoglein 3 Order and Dynamics in Desmosomes Determined by Fluorescence Polarization Microscopy. Biophysical journal\| PubMed ID: 29212005	2017
Mapping the 3D orientation of piconewton integrin traction forces. Nature methods\| PubMed ID: 29256495	2018
Bridging the gap: Super-resolution microscopy of epithelial cell junctions. Tissue barriers\| PubMed ID: 29420122	2018
Cutting Edge: Intracellular IFN-β and Distinct Type I IFN Expression Patterns in Circulating Systemic Lupus Erythematosus B Cells. Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 30201809	2018
The desmosome is a mesoscale lipid raft-like membrane domain. Molecular biology of the cell\| PubMed ID: 30943110	2019
Pharmacologic inhibition of LIMK1 provides dendritic spine resilience against β-amyloid. Science signaling\| PubMed ID: 31239325	2019
DNA mechanotechnology reveals that integrin receptors apply pN forces in podosomes on fluid substrates. Nature communications\| PubMed ID: 31628308	2019
Variable incidence angle linear dichroism (VALiD): a technique for unique 3D orientation measurement of fluorescent ensembles. Optics express\| PubMed ID: 32225599	2020
Protein exchange is reduced in calcium-independent epithelial junctions. The Journal of cell biology\| PubMed ID: 32399559	2020
EGFR activation attenuates the mechanical threshold for integrin tension and focal adhesion formation. Journal of cell science\| PubMed ID: 32546532	2020
Live-cell super-resolved PAINT imaging of piconewton cellular traction forces. Nature methods\| PubMed ID: 32929270	2020
dSTORM Imaging and Analysis of Desmosome Architecture. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 33225407	2021
Novel role of the dietary flavonoid fisetin in suppressing rRNA biogenesis. Laboratory investigation; a journal of technical methods and pathology\| PubMed ID: 34267320	2021
Turn-key mapping of cell receptor force orientation and magnitude using a commercial structured illumination microscope. Nature communications\| PubMed ID: 34344862	2021
Author Correction: Turn-key mapping of cell receptor force orientation and magnitude using a commercial structured illumination microscope. Nature communications\| PubMed ID: 34531385	2021
Desmosomes undergo dynamic architectural changes during assembly and maturation. Tissue barriers\| PubMed ID: 34983311	2022

Alexa L. Mattheyses

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