Single-Molecule Fingerprinting Reveals Different Growth Mechanisms in Seed Amplification Assays for Different Polymorphs of α-Synuclein Fibrils.

α-Synuclein (αSyn) aggregates, detected in the biofluids of patients with Parkinson's disease (PD), have the ability to catalyze their own aggregation, leading to an increase in the number and size of aggregates. This self-templated amplification is used by newly developed assays to diagnose Parkinson's disease and turns the presence of αSyn aggregates into a biomarker of the disease. It has become evident that αSyn can form fibrils with slightly different structures, called "strains" or polymorphs, but little is known about their differential reactivity in diagnostic assays.

Mechanoimmunology in the solid tumor microenvironment.

The tumor microenvironment (TME) is a complex and dynamic ecosystem that adjoins the cancer cells within solid tumors and comprises distinct components such as extracellular matrix, stromal and immune cells, blood vessels, and an abundance of signaling molecules. In recent years, the mechanical properties of the TME have emerged as critical determinants of tumor progression and therapeutic response. Aberrant mechanical cues, including altered tissue architecture and stiffness, contribute to tumor progression, metastasis, and resistance to treatment.

A hepatic network of dendritic cells mediates CD4 T cell help outside lymphoid organs.

While CD4 T cells are a prerequisite for CD8 T cell-mediated protection against intracellular hepatotropic pathogens, the mechanisms facilitating the transfer of CD4-help to intrahepatic CD8 T cells are unknown. Here, we developed an experimental system to investigate cognate CD4 and CD8 T cell responses to a model-antigen expressed de novo in hepatocytes and reveal that after initial priming, effector CD4 and CD8 T cells migrate into portal tracts and peri-central vein regions of the liver where they cluster with type-1 conventional dendritic cells. These dendritic cells are locally licensed by CD4 T cells and expand the number of CD8 T cells in situ, resulting in larger effector and memory CD8 T cell pools.

Chemokine receptor distribution on the surface of repolarizing T cells.

T cells migrate constitutively with a polarized morphology, underpinned by signaling compartmentalization and discrete cytoskeletal organizations, giving rise to a dynamic and expansive leading edge, distinct from the stable and constricted uropod at the rear. In vivo, the motion and function of T cells at various stages of differentiation is highly directed by chemokine gradients. When cognate ligands bind chemokine receptors on their surface, T cells respond by reorientating their polarity axis and migrating toward the source of the chemokine signal.

Programming of Multicellular Patterning with Mechano-Chemically Microstructured Cell Niches.

Multicellular patterning of stem-cell-derived tissue models is commonly achieved via self-organizing activities triggered by exogenous morphogenetic stimuli. However, such tissue models are prone to stochastic behavior, limiting the reproducibility of cellular composition and forming non-physiological architectures. To enhance multicellular patterning in stem cell-derived tissues, a method for creating complex tissue microenvironments endowed with programmable multimodal mechano-chemical cues, including conjugated peptides, proteins, morphogens, and Young's moduli defined over a range of stiffnesses is developed.

T cell cytoskeletal forces shape synapse topography for targeted lysis via membrane curvature bias of perforin.

Cytotoxic T lymphocytes (CTLs) lyse target cells by delivering lytic granules that contain the pore former perforin to the cytotoxic immunological synapse. Here, we establish that opposing cytoskeletal forces drive lytic granule polarization and simultaneously shape T cell synapse topography to enhance target perforation. At the cell rear, actomyosin contractility drives the anterograde movement of lytic granules toward the nucleus.

T cell morphodynamics reveal periodic shape oscillations in three-dimensional migration.

T cells use sophisticated shape dynamics (morphodynamics) to migrate towards and neutralize infected and cancerous cells. However, there is limited quantitative understanding of the migration process in three-dimensional extracellular matrices (ECMs) and across timescales. Here, we leveraged recent advances in lattice light-sheet microscopy to quantitatively explore the three-dimensional morphodynamics of migrating T cells at high spatio-temporal resolution.

SNX9-induced membrane tubulation regulates CD28 cluster stability and signalling.

T cell activation requires engagement of a cognate antigen by the T cell receptor (TCR) and the co-stimulatory signal of CD28. Both TCR and CD28 aggregate into clusters at the plasma membrane of activated T cells. While the role of TCR clustering in T cell activation has been extensively investigated, little is known about how CD28 clustering contributes to CD28 signalling.

4D analysis of malaria parasite invasion offers insights into erythrocyte membrane remodeling and parasitophorous vacuole formation.

Host membrane remodeling is indispensable for viruses, bacteria, and parasites, to subvert the membrane barrier and obtain entry into cells. The malaria parasite Plasmodium spp. induces biophysical and molecular changes to the erythrocyte membrane through the ordered secretion of its apical organelles.

Cytotoxic T cells swarm by homotypic chemokine signalling.

Cytotoxic T lymphocytes (CTLs) are thought to arrive at target sites either via random search or following signals by other leukocytes. Here, we reveal independent emergent behaviour in CTL populations attacking tumour masses. Primary murine CTLs coordinate their migration in a process reminiscent of the swarming observed in neutrophils.

The Lifeact-EGFP mouse is a translationally controlled fluorescent reporter of T cell activation.

It has become increasingly evident that T cell functions are subject to translational control in addition to transcriptional regulation. Here, by using live imaging of CD8 T cells isolated from the Lifeact-EGFP mouse, we show that T cells exhibit a gain in fluorescence intensity following engagement of cognate tumour target cells. The GFP signal increase is governed by Erk1/2-dependent distal T cell receptor (TCR) signalling and its magnitude correlates with IFN-γ and TNF-α production, which are hallmarks of T cell activation.

Brightness-gated two-color coincidence detection unravels two distinct mechanisms in bacterial protein translation initiation.

Life on the molecular scale is based on a complex interplay of biomolecules under which the ability of binding is crucial. Fluorescence based two-color coincidence detection (TCCD) is commonly used to characterize molecular binding, but suffers from an underestimation of coincident events. Here, we introduce a brightness-gated TCCD which overcomes this limitation and benchmark our approach with two custom-made calibration samples.

Flotillins promote T cell receptor sorting through a fast Rab5-Rab11 endocytic recycling axis.

The targeted endocytic recycling of the T cell receptor (TCR) to the immunological synapse is essential for T cell activation. Despite this, the mechanisms that underlie the sorting of internalised receptors into recycling endosomes remain poorly understood. To build a comprehensive picture of TCR recycling during T cell activation, we developed a suite of new imaging and quantification tools centred on photoactivation of fluorescent proteins.

Impact of Molecular Crowding on Translational Mobility and Conformational Properties of Biological Macromolecules.

Effects of molecular crowding on structural and dynamical properties of biological macromolecules do depend on the concentration of crowding agents but also on the molecular mass and the structural compactness of the crowder molecules. By employing fluorescence correlation spectroscopy (FCS), we investigated the translational mobility of several biological macromolecules ranging from 17 kDa to 2.7 MDa.

Mechanoimmunology: molecular-scale forces govern immune cell functions.

Immune cell recognition of antigens is a pivotal process in initiating immune responses against injury, pathogens, and cancers. Breakthroughs over the past decade support a major role for mechanical forces in immune responses, laying the foundation for the emerging field of mechanoimmunology. In this Perspective, we discuss the mechanical forces acting at the level of ligand-receptor interactions and how they underpin receptor triggering, signal initiation, and immune cell activation.

Single-Molecule FRET Measurements in Additive-Enriched Aqueous Solutions.

The addition of high amounts of chemical denaturants, salts, viscosity enhancers or macro-molecular crowding agents has an impact on the physical properties of buffer solutions. Among others, the (microscopic) viscosity, the refractive index, the dielectric constant, and the ionic strength can be affected. Here, we systematically evaluate the importance of solvent characteristics with respect to single-molecule FRET (smFRET) data.

Accurate fluorescence quantum yield determination by fluorescence correlation spectroscopy.

Here, we present a comparative method for the accurate determination of fluorescence quantum yields (QYs) by fluorescence correlation spectroscopy. By exploiting the high sensitivity of single-molecule spectroscopy, we obtain the QYs of samples in the microliter range and at (sub)nanomolar concentrations. Additionally, in combination with fluorescence lifetime measurements, our method allows the quantification of both static and collisional quenching constants.

Inter-dye distance distributions studied by a combination of single-molecule FRET-filtered lifetime measurements and a weighted accessible volume (wAV) algorithm.

Förster resonance energy transfer (FRET) is an important tool for studying the structural and dynamical properties of biomolecules. The fact that both the internal dynamics of the biomolecule and the movements of the biomolecule-attached dyes can occur on similar timescales of nanoseconds is an inherent problem in FRET studies. By performing single-molecule FRET-filtered lifetime measurements, we are able to characterize the amplitude of the motions of fluorescent probes attached to double-stranded DNA standards by means of flexible linkers.

Conformational state distributions and catalytically relevant dynamics of a hinge-bending enzyme studied by single-molecule FRET and a coarse-grained simulation.

Over the last few decades, a view has emerged showing that multidomain enzymes are biological machines evolved to harness stochastic kicks of solvent particles into highly directional functional motions. These intrinsic motions are structurally encoded, and Nature makes use of them to catalyze chemical reactions by means of ligand-induced conformational changes and states redistribution. Such mechanisms align reactive groups for efficient chemistry and stabilize conformers most proficient for catalysis.

Nanosecond dynamics of calmodulin and ribosome-bound nascent chains studied by time-resolved fluorescence anisotropy.

We report a time-resolved fluorescence anisotropy study of ribosome-bound nascent chains (RNCs) of calmodulin (CaM), a prototypical member of the EF-hand family of calcium-sensing proteins. As shown in numerous studies, in vitro protein refolding can differ substantially from biosynthetic protein folding, which takes place cotranslationally and depends on the rate of polypeptide chain elongation. A challenge in this respect is to characterize the adopted conformations of nascent chains before their release from the ribosome.