The αβ T-cell receptor (TCR) on each T lymphocyte mediates exquisite specificity for a particular foreign peptide bound to a major histocompatibility complex molecule (pMHC) displayed on the surface of altered cells. This recognition stimulates protection in the mammalian host against intracellular pathogens, including viruses, and involves piconewton forces that accompany pMHC ligation. Physical forces are generated by T-lymphocyte movement during immune surveillance as well as by cytoskeletal rearrangements at the immunological synapse following cessation of cell migration. The mechanistic explanation for how TCRs distinguish between foreign and self-peptides bound to a given MHC molecule is unclear: peptide residues themselves comprise few of the TCR contacts on the pMHC, and pathogen-derived peptides are scant among myriad self-peptides bound to the same MHC class arrayed on infected cells. Using optical tweezers and DNA tether spacer technology that permit piconewton force application and nanometer scale precision, we have determined how bioforces relate to self versus nonself discrimination. Single-molecule analyses involving isolated αβ-heterodimers as well as complete TCR complexes on T lymphocytes reveal that the FG loop in the β-subunit constant domain allosterically controls both the variable domain module's catch bond lifetime and peptide discrimination via force-driven conformational transition. In contrast to integrins, the TCR interrogates its ligand via a strong force-loaded state with release through a weakened, extended state. Our work defines a key element of TCR mechanotransduction, explaining why the FG loop structure evolved for adaptive immunity in αβ but not γδTCRs or immunoglobulins.mechanosensor | T-cell receptor | peptide discrimination | optical tweezers | catch bond A ntigen recognition by T lymphocytes is a crucial feature of adaptive immunity. This process requires the interaction of a clone-specific T-cell receptor (TCR) via its membrane distal variable module with a cognate peptide bound to a major histocompatibility complex molecule (pMHC) (refs. 1 and 2 and references therein). "Foreign" peptide antigens derived from infectious or other cell-altering processes including oncogenic transformation are presented either on a surface of the perturbed cell directly or indirectly via cross-presentation on antigen-presenting cells (APC). In either case, ligation of the relevant TCRαβ heterodimer initiates a cascade of T-cell signaling events following exposure of the immunoreceptor tyrosine-based activation motif (ITAM) elements in the cytoplasmic tail of the noncovalently associated subunits (CD3eγ, CD3eδ, and CD3ζζ) composing the TCR complex in 1:1:1:1 dimer stoichiometry. This accessibility allows the active kinase, Lck, to bind and phosphorylate ITAMs followed by recruitment and activation of a second tyrosine kinase, ZAP-70 (3-6). In turn, multiple downstream pathways are engaged, including transcriptional regulators controlling activation and differentiation of T cells (7,8). Thym...
Cellobiohydrolase 1 from Trichoderma reesei (TrCel7A) processively hydrolyses cellulose into cellobiose. Although enzymatic techniques have been established as promising tools in biofuel production, a clear understanding of the motor's mechanistic action has yet to be revealed. Here, we develop an optical tweezers-based single-molecule (SM) motility assay for precision tracking of TrCel7A. Direct observation of motility during degradation reveals processive runs and distinct steps on the scale of 1 nm. Our studies suggest TrCel7A is not mechanically limited, can work against 20 pN loads and speeds up when assisted. Temperature-dependent kinetic studies establish the energy requirements for the fundamental stepping cycle, which likely includes energy from glycosidic bonds and other sources. Through SM measurements of isolated TrCel7A domains, we determine that the catalytic domain alone is sufficient for processive motion, providing insight into TrCel7A's molecular motility mechanism.
The cytoskeleton provides structure and plays an important role in cellular function such as migration, resisting compression forces, and transport. The cytoskeleton also reacts to physical cues such as fluid shear stress or extracellular matrix remodeling by reorganizing filament associations, most commonly focal adhesions and cell-cell cadherin junctions. These mechanical stimuli can result in genome-level changes, and the physical connection of the cytoskeleton to the nucleus provides an optimal conduit for signal transduction by interfacing with nuclear envelope proteins, called nesprins, within the LINC (linker of the nucleus to the cytoskeleton) complex. Using single-molecule on single nuclei assays, we report that the interactions between the nucleus and the cytoskeleton, thought to be nesprin-cytoskeleton interactions, are highly sensitive to force magnitude and direction depending on whether cells are historically interfaced with the matrix or with cell aggregates. Application of ∼10-30 pN forces to these nesprin linkages yielded structural transitions, with a base transition size of 5-6 nm, which are speculated to be associated with partial unfoldings of the spectrin domains of the nesprins and/or structural changes of histones within the nucleus.
Cellulose biosynthesis in sessile bacterial colonies originates in the membrane-integrated bacterial cellulose synthase (Bcs) AB complex. We utilize optical tweezers to measure single-strand cellulose biosynthesis by BcsAB from Rhodobacter sphaeroides . Synthesis depends on uridine diphosphate glucose, Mg 2+ , and cyclic diguanosine monophosphate, with the last displaying a retention time of ∼80 min. Below a stall force of 12.7 pN, biosynthesis is relatively insensitive to force and proceeds at a rate of one glucose addition every 2.5 s at room temperature, increasing to two additions per second at 37°. At low forces, conformational hopping is observed. Single-strand cellulose stretching unveiled a persistence length of 6.2 nm, an axial stiffness of 40.7 pN, and an ability for complexes to maintain a tight grip, with forces nearing 100 pN. Stretching experiments exhibited hysteresis, suggesting that cellulose microstructure underpinning robust biofilms begins to form during synthesis. Cellohexaose spontaneously binds to nascent single cellulose strands, impacting polymer mechanical properties and increasing BcsAB activity.
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