Small, Dynamic Clusters of Tir-Intimin Seed Actin Polymerization.

The understanding of actin pedestal formation by enteropathogenic Escherichia coli (EPEC) relies mainly on static ensemble information obtained from cell lysates. Here, the dynamic nature of signaling components on the subsecond timescale, which resemble phase condensates, is demonstrated. Unlike in vitro phase condensates, transfected intimin receptor (Tir) and downstream component form clusters 200 nm in diameter that are spaced ≈500 nm on average, indicating cellular regulation. On supported lipid bilayers with diffusive intimin, Tir-expressing fibroblasts formed Tir-intimin clusters even without Tir tyrosines, although Tir tyrosine phosphorylation is necessary for actin polymerization from clusters. Single-molecule tracking showed that Tir is diffusive in the clusters and exchanges with Tir in the plasma membrane. Further, Nck and N-WASP bind to the clusters and exchange with cytoplasmic molecules. Tir has a similar cluster lifetime to Nck, but longer than that of N-WASP. Actin polymerization from the clusters requires N-WASP binding, involved Arp2/3 activation, and stabilized N-WASP clusters. These dynamic properties are distinct from larger in vitro systems and do not depend significantly upon crosslinking. Thus, Tir-intimin clusters in the plasma membrane are limited in size by exchange and enhance signaling needed for actin polymerization that enables strong and stable bacterial attachment to host cells.

Competition for shared downstream signaling molecules establishes indirect negative feedback between EGFR and EphA2.

Cells sense a variety of extracellular growth factors and signaling molecules through numerous distinct receptor tyrosine kinases (RTKs) on the cell surface. In many cases, the same intracellular signaling molecules interact with more than one type of RTK. How signals from different RTKs retain the identity of the triggering receptor and how (or if) different receptors may synergize or compete remain largely unknown. Here we utilize an experimental strategy, combining microscale patterning and single-molecule imaging, to measure the competition between ephrin-A1:EphA2 and epidermal growth factor (EGF):EGF receptor (EGFR) ligand-receptor complexes for the shared downstream signaling molecules, Grb2 and SOS. The results reveal a distinct hierarchy, in which newly formed EGF:EGFR complexes outcompete ephrin-A1:EphA2 for Grb2 and SOS, revealing a type of negative crosstalk interaction fundamentally controlled by chemical mass action and protein copy number limitations.

Probing the effect of clustering on EphA2 receptor signaling efficiency by subcellular control of ligand-receptor mobility.

Clustering of ligand:receptor complexes on the cell membrane is widely presumed to have functional consequences for subsequent signal transduction. However, it is experimentally challenging to selectively manipulate receptor clustering without altering other biochemical aspects of the cellular system. Here, we develop a microfabrication strategy to produce substrates displaying mobile and immobile ligands that are separated by roughly 1 µm, and thus experience an identical cytoplasmic signaling state, enabling precision comparison of downstream signaling reactions. Applying this approach to characterize the ephrinA1:EphA2 signaling system reveals that EphA2 clustering enhances both receptor phosphorylation and downstream signaling activity. Single-molecule imaging clearly resolves increased molecular binding dwell times at EphA2 clusters for both Grb2:SOS and NCK:N-WASP signaling modules. This type of intracellular comparison enables a substantially higher degree of quantitative analysis than is possible when comparisons must be made between different cells and essentially eliminates the effects of cellular response to ligand manipulation.

Nanopore-mediated protein delivery enabling three-color single-molecule tracking in living cells.

Multicolor single-molecule tracking (SMT) provides a powerful tool to mechanistically probe molecular interactions in living cells. However, because of the limitations in the optical and chemical properties of currently available fluorophores and the multiprotein labeling strategies, intracellular multicolor SMT remains challenging for general research studies. Here, we introduce a practical method employing a nanopore-electroporation (NanoEP) technique to deliver multiple organic dye-labeled proteins into living cells for imaging. It can be easily expanded to three channels in commercial microscopes or be combined with other in situ labeling methods. Utilizing NanoEP, we demonstrate three-color SMT for both cytosolic and membrane proteins. Specifically, we simultaneously monitored single-molecule events downstream of EGFR signaling pathways in living cells. The results provide detailed resolution of the spatial localization and dynamics of Grb2 and SOS recruitment to activated EGFR along with the resultant Ras activation.

Spatially modulated ephrinA1:EphA2 signaling increases local contractility and global focal adhesion dynamics to promote cell motility.

Recent studies have revealed pronounced effects of the spatial distribution of EphA2 receptors on cellular response to receptor activation. However, little is known about molecular mechanisms underlying this spatial sensitivity, in part due to lack of experimental systems. Here, we introduce a hybrid live-cell patterned supported lipid bilayer experimental platform in which the sites of EphA2 activation and integrin adhesion are spatially controlled. Using a series of live-cell imaging and single-molecule tracking experiments, we map the transmission of signals from ephrinA1:EphA2 complexes. Results show that ligand-dependent EphA2 activation induces localized myosin-dependent contractions while simultaneously increasing focal adhesion dynamics throughout the cell. Mechanistically, Src kinase is activated at sites of ephrinA1:EphA2 clustering and subsequently diffuses on the membrane to focal adhesions, where it up-regulates FAK and paxillin tyrosine phosphorylation. EphrinA1:EphA2 signaling triggers multiple cellular responses with differing spatial dependencies to enable a directed migratory response to spatially resolved contact with ephrinA1 ligands.

Asymmetric polar localization dynamics of the serine chemoreceptor protein Tsr in Escherichia coli.

The spatial location of proteins in living cells can be critical for their function. For example, the E. coli chemotaxis machinery is localized to the cell poles. Here we describe the polar localization of the serine chemoreceptor Tsr using a strain synthesizing a fluorescent Tsr-Venus fusion at a low level from a single-copy chromosomal construct. Using photobleaching and imaging during recovery by new synthesis, we observed distinct asymmetry between a bright (old) pole and a dim (new) pole. The old pole was shown to be a more stable cluster and to recover after photobleaching faster, which is consistent with the hypothesis that newly synthesized Tsr proteins are inserted directly at or near the old pole. The new pole was shown to be a less stable cluster and to exchange proteins freely with highly mobile Tsr-Venus proteins diffusing in the membrane. We propose that the new pole arises from molecules escaping from the old pole and diffusing to the new pole where a more stable cluster forms over time. Our localization imaging data support a model in which a nascent new pole forms prior to stable cluster formation.

EGFR family and Src family kinase interactions: mechanics matters?

Receptor tyrosine kinases (RTKs), such as the EGF receptor family, and adhesion molecules, such as integrins, have historically been viewed to have distinctly separable roles in the cell. In this classical view, integrins mediate mechanical interactions between the cell and its surrounding extracellular matrix while RTKs handle signaling to modulate cellular behavior. Although crosstalk between these receptor pathways has been known to exist for a long time, this has generally been attributed to effects significantly downstream from the receptors themselves. In recent years, however, EGFR family RTKs have been found to directly participate in integrin-mediated force sensing, revealing a more complex interplay among these cellular components than originally appreciated. Here we briefly review the classical understanding of EGFR family RTK signaling and then provide a broadened perspective based on recent results.

Real-Time Single Molecule Visualization of SH2 Domain Membrane Recruitment in Growth Factor Stimulated Cells.

In the last decade, single molecule tracking (SMT) techniques have emerged as a versatile tool for molecular cell biology research. This approach allows researchers to monitor the real-time behavior of individual molecules in living cells with nanometer and millisecond resolution. As a result, it is possible to visualize biological processes as they occur at a molecular level in real time. Here we describe a method for the real-time visualization of SH2 domain membrane recruitment from the cytoplasm to epidermal growth factor (EGF) induced phosphotyrosine sites on the EGF receptor. Further, we describe methods that utilize SMT data to define SH2 domain membrane dynamics parameters such as binding (τ), dissociation (k d), and diffusion (D) rates. Together these methods may allow us to gain greater understanding of signal transduction dynamics and the molecular basis of disease-related aberrant pathways.

Time-resolved multimodal analysis of Src Homology 2 (SH2) domain binding in signaling by receptor tyrosine kinases.

While the affinities and specificities of SH2 domain-phosphotyrosine interactions have been well characterized, spatio-temporal changes in phosphosite availability in response to signals, and their impact on recruitment of SH2-containing proteins in vivo, are not well understood. To address this issue, we used three complementary experimental approaches to monitor phosphorylation and SH2 binding in human A431 cells stimulated with epidermal growth factor (EGF): 1) phospho-specific mass spectrometry; 2) far-Western blotting; and 3) live cell single-molecule imaging of SH2 membrane recruitment. Far-Western and MS analyses identified both well-established and previously undocumented EGF-dependent tyrosine phosphorylation and binding events, as well as dynamic changes in binding patterns over time. In comparing SH2 binding site phosphorylation with SH2 domain membrane recruitment in living cells, we found in vivo binding to be much slower. Delayed SH2 domain recruitment correlated with clustering of SH2 domain binding sites on the membrane, consistent with membrane retention via SH2 rebinding.

Macrophage migration inhibitory factor down-regulates the RANKL-RANK signaling pathway by activating Lyn tyrosine kinase in mouse models.

OBJECTIVE: Macrophage migration inhibitory factor (MIF) is an important modulator of innate and adaptive immunity as well as local inflammatory responses. We previously reported that MIF down-regulated osteoclastogenesis through a mechanism that requires CD74. The aim of the current study was to examine whether MIF modulates osteoclastogenesis through Lyn phosphorylation, and whether down-regulation of RANKL-mediated signaling requires the association of CD74, CD44, and Lyn. METHODS: CD74-knockout (CD74-KO), CD44-KO, and Lyn-KO mouse models were used to investigate whether Lyn requires these receptors and coreceptors. The effects of MIF on osteoclastogenesis were assessed using Western blot analysis, small interfering RNA (siRNA)-targeted down-regulation of Lyn, Lyn-KO mice, and real-time imaging of Lyn molecules to surface proteins. RESULTS: MIF treatment induced Lyn expression, and MIF down-regulated RANKL-induced activator protein 1 (AP-1) and the Syk/phospholipase Cγ cascade during osteoclastogenesis through activated Lyn tyrosine kinase. The results of immunoprecipitation studies revealed that MIF receptors associated with Lyn in response to MIF treatment. Studies using Lyn-specific siRNA and Lyn-KO mice confirmed our findings. CONCLUSION: Our findings indicate that the tyrosine kinase Lyn is activated when MIF binds to its receptor CD74 and its coreceptor CD44 and, in turn, down-regulates the RANKL-mediated signaling cascade by suppressing NF-ATc1 protein expression through down-regulation of AP-1 and calcium signaling components.

Dynamics of the serine chemoreceptor in the Escherichia coli inner membrane: a high-speed single-molecule tracking study.

We investigated the mobility of the polar localized serine chemoreceptor, Tsr, labeled by the fluorescent protein Venus in the inner membrane of live Escherichia coli cells at observation rates up to 1000 Hz. A fraction (7%) of all Tsr molecules shows free diffusion over the entire cell surface with an average diffusion coefficient of 0.40 ± 0.01 µm(2) s(-1). The remaining molecules were found to be ultimately confined in compartments of size 290 ± 15 nm and showed restricted diffusion at an inner barrier found at 170 ± 10 nm. At the shortest length-scales (<170 nm), all Tsr molecules diffuse equally. Disruption of the cytoskeleton and rounding of the cells resulted in an increase in the mobile fraction of Tsr molecules and a fragmenting of the previously polar cluster of Tsr consistent with a curvature-based mechanism of Tsr cluster maintenance.

Fast rebinding increases dwell time of Src homology 2 (SH2)-containing proteins near the plasma membrane.

Receptor tyrosine kinases (RTKs) control a host of biological functions by phosphorylating tyrosine residues of intracellular proteins upon extracellular ligand binding. The phosphotyrosines (p-Tyr) then recruit a subset of ∼100 Src homology 2 (SH2) domain-containing proteins to the cell membrane. The in vivo kinetics of this process are not well understood. Here we use total internal reflection (TIR) microscopy and single-molecule imaging to monitor interactions between SH2 modules and p-Tyr sites near the cell membrane. We found that the dwell time of SH2 modules within the TIR illumination field is significantly longer than predictions based on chemical dissociation rate constants, suggesting that SH2 modules quickly rebind to nearby p-Tyr sites after dissociation. We also found that, consistent with the rebinding model, the effective diffusion constant is negatively correlated with the respective dwell time for different SH2 domains and the dwell time is positively correlated with the local density of RTK phosphorylation. These results suggest a mechanism whereby signal output can be regulated through the spatial organization of multiple binding sites, which will prompt reevaluation of many aspects of RTK signaling, such as signaling specificity, mechanisms of spatial control, and noise suppression.

Gyromagnetic imaging: dynamic optical contrast using gold nanostars with magnetic cores.

Plasmon-resonant nanoparticles with optical scattering in the near-infrared (NIR) are valuable contrast agents for biophotonic imaging and may be detected at the single-particle limit against a dark background, but their contrast is often limited in environments with high noise. Here we consider gyromagnetic imaging as a dynamic mode of optical contrast, using gold nanostars with superparamagnetic cores. The nanostars exhibit polarization-sensitive NIR scattering and can produce a frequency-modulated signal in response to a rotating magnetic field gradient. This periodic "twinkling" can be converted into Fourier-domain images with a dramatic reduction in background. We demonstrate gyromagnetic imaging of nanostars inside of tumor cells, using broadband excitation: while their time-domain signals are obscured by incoherent scattering, their Fourier-domain signals can be clearly resolved in less than a second. The gyromagnetically active nanostars do not cause a loss in viability, and can even have a mild stimulatory effect on cell growth.