Alexander Gondarenko

Cells test substrate rigidity by local contractions on submicrometer pillars

Cell growth and differentiation are critically dependent upon matrix rigidity, yet many aspects of the cellular rigidity-sensing mechanism are not understood. Here, we analyze matrix forces after initial cell–matrix contact, when early rigidity-sensing events occur, using a series of elastomeric pillar arrays with dimensions extending to the submicron scale (2, 1, and 0.5 μm in diameter covering a range of stiffnesses). We observe that the cellular response is fundamentally different on micron-scale and submicron pillars. On 2-μm diameter pillars, adhesions form at the pillar periphery, forces are directed toward the center of the cell, and a constant maximum force is applied independent of stiffness. On 0.5-μm diameter pillars, adhesions form on the pillar tops, and local contractions between neighboring pillars are observed with a maximum displacement of ∼60 nm, independent of stiffness. Because mutants in rigidity sensing show no detectable displacement on 0.5-μm diameter pillars, there is a correlation between local contractions to 60 nm and rigidity sensing. Localization of myosin between submicron pillars demonstrates that submicron scale myosin filaments can cause these local contractions. Finally, submicron pillars can capture many details of cellular force generation that are missed on larger pillars and more closely mimic continuous surfaces.

Radio frequency electrical transduction of graphene mechanical resonators

We report radio frequency (rf) electrical readout of graphene mechanical resonators. The mechanical motion is actuated and detected directly by using a vector network analyzer, employing a local gate to minimize parasitic capacitance. A resist-free doubly clamped sample with resonant frequency ∼34 MHz, quality factor ∼10 000 at 77 K, and signal-to-background ratio of over 20 dB is demonstrated. In addition to being over two orders of magnitude faster than the electrical rf mixing method, this technique paves the way for use of graphene in rf devices such as filters and oscillators.

CD28 and CD3 have complementary roles in T-cell traction forces

Significance Cells have the remarkable ability to sense the mechanical properties of the extracellular environment. This has been developed primarily in the context of cell interaction with extracellular matrix through integrins, but recent studies showed that T cells exhibit mechanosensing through CD3 and CD28, nonintegrin receptors that provide activation and costimulatory signals. This report demonstrates that T cells generate significant forces through the T-cell receptor (TCR) and CD28, and delineates the contributions of each in force generation. Moreover, the distribution of force generation and local assembly of active signaling intermediates suggest similarities between TCR- and integrin-based connections between the cell and substrate. Mechanical forces have key roles in regulating activation of T cells and coordination of the adaptive immune response. A recent example is the ability of T cells to sense the rigidity of an underlying substrate through the T-cell receptor (TCR) coreceptor CD3 and CD28, a costimulation signal essential for cell activation. In this report, we show that these two receptor systems provide complementary functions in regulating the cellular forces needed to test the mechanical properties of the extracellular environment. Traction force microscopy was carried out on primary human cells interacting with micrometer-scale elastomer pillar arrays presenting activation antibodies to CD3 and/or CD28. T cells generated traction forces of 100 pN on arrays with both antibodies. By providing one antibody or the other in solution instead of on the pillars, we show that force generation is associated with CD3 and the TCR complex. Engagement of CD28 increases traction forces associated with CD3 through the signaling pathway involving PI3K, rather than providing additional coupling between the cell and surface. Force generation is concentrated to the cell periphery and associated with molecular complexes containing phosphorylated Pyk2, suggesting that T cells use processes that share features with integrin signaling in force generation. Finally, the ability of T cells to apply forces through the TCR itself, rather than the CD3 coreceptor, was tested. Mouse cells expressing the 5C.C7 TCR exerted traction forces on pillars presenting peptide-loaded MHCs that were similar to those with α-CD3, suggesting that forces are applied to antigen-presenting cells during activation.

Electrically integrated SU-8 clamped graphene drum resonators for strain engineering

Graphene mechanical resonators are the ultimate two-dimensional nanoelectromechanical systems (NEMS) with applications in sensing and signal processing. While initial devices have shown promising results, an ideal graphene NEMS resonator should be strain engineered, clamped at the edge without trapping gas underneath, and electrically integratable. In this letter, we demonstrate fabrication and direct electrical measurement of circular SU-8 polymer-clamped chemical vapor deposition (CVD) graphene drum resonators. The clamping increases device yield and responsivity, while providing a cleaner resonance spectrum from eliminated edge modes. Furthermore, this resonator is highly strained, indicating its potential in strain engineering for performance enhancement.Graphene mechanical resonators are the ultimate two-dimensional nanoelectromechanical systems (NEMS) with applications in sensing and signal processing. While initial devices have shown promising results, an ideal graphene NEMS resonator should be strain engineered, clamped at the edge without trapping gas underneath, and electrically integratable. In this Letter, we demonstrate fabrication and direct electrical measurement of circular SU-8 polymer-clamped chemical vapor deposition graphene drum resonators. The clamping increases device yield and responsivity, while providing a cleaner resonance spectrum from eliminated edge modes. Furthermore, the clamping induces a large strain in the resonator, increasing its resonant frequency.

α-Actinin links extracellular matrix rigidity-sensing contractile units with periodic cell-edge retractions

During cell migration, the cell edge undergoes periodic protrusion–retraction cycles. Quantitative analyses of the forces at the cell edge that drive these cycles are provided. We show that α-actinin links local contractile units and the global actin flow forces at the cell edge and present a novel model based on these results.

High-resolution imaging of the immunological synapse and T-cell receptor microclustering through microfabricated substrates

T-cell activation via antigen presentation is associated with the formation of a macromolecular membrane assembly termed the immunological synapse (IS). The genesis of the IS and the onset of juxtacrine signalling is characterized by the formation of cell membrane microclusters and the organization of such into segregated microdomains. A central zone rich in T-cell receptor (TCR)–major histocompatibility complex microclusters termed the central supramolecular activation cluster (cSMAC) forms the bullseye of this structure, while the cellular interface surrounding the cSMAC is characterized by regions enriched in adhesion and co-stimulatory molecules. In vitro, the study of dynamic TCR microcluster coalescence and IS genesis in T-cell populations is hampered by cell migration within the culture system and resolution constraints resulting from lateral cell–cell contact. Here, we detail a novel system describing the fabrication of micropit arrays designed to sequester single T-cell–antigen presenting cell (APC) conjugates and promote IS formation in the horizontal imaging plane for high-resolution studies of microcluster dynamics. We subsequently use this system to describe the formation of the cSMAC in T-cell populations and to investigate the morphology of the interfacial APC membrane.

Graphene metallization of high-stress silicon nitride resonators for electrical integration

High stress stoichiometric silicon nitride resonators, whose quality factors exceed one million, have shown promise for applications in sensing, signal processing, and optomechanics. Yet, electrical integration of the insulating silicon nitride resonators has been challenging, as depositing even a thin layer of metal degrades the quality factor significantly. In this work, we show that graphene used as a conductive coating for Si3N4 membranes reduces the quality factor by less than 30% on average, which is minimal when compared to the effect of conventional metallization layers such as chromium or aluminum. The electrical integration of Si3N4-Graphene (SiNG) heterostructure resonators is demonstrated with electrical readout and electrostatic tuning of the frequency by up to 0.3% per volt. These studies demonstrate the feasibility of hybrid graphene/nitride mechanical resonators in which the electrical properties of graphene are combined with the superior mechanical performance of silicon nitride.

Modulation of mechanical resonance by chemical potential oscillation in graphene

By coupling to electrons in the quantum Hall regime, the mechanical response of graphene resonators is modulated by changes in the chemical potential.

T-Cell Receptor Activation Initiates Multiple Modes of Actin Polymerization within the Immune Synapse

The immune synapse is a key point of communication between T cells and antigen presenting cells. The layout of this interface is driven in large part by a complex cytoskeletal structure. This report focuses on modulation of the cytoskeleton by T Cell Receptor (TCR) signaling using micropatterned surfaces presenting OKT3 (an antibody that activates the TCR signaling component CD3) and ICAM-1 to T cells. Previous studies by our group demonstrated that micro-scale features of OKT3 (red, top row) induce lamellipodial polymerization (white arrows) of actin (green) on surrounding regions of ICAM-1. We show here a second type of polymerization, the formation of linear, consolidated F-actin structures from sites of CD3 engagement (blue arrow), resembling actin comets observed in other systems. This mode of actin polymerization occurs concurrent to lamellipodium extension. Furthermore, polymerization is initiated by sharp, external corners of angular patterns of OKT3 (bottom row). This latter result suggests mechanical forces, which are concentrated at such corners under cellular tension, initiate this modality of actin polymerization. The different contributions of these behaviors on immune synapse function remain to be determined.View Large Image | View Hi-Res Image | Download PowerPoint Slide