An interpretation of fluctuations in enzyme catalysis rate, spectral diffusion, and radiative component of lifetimes in terms of electric field fluctuations

Time-dependent fluctuations in the catalysis rate ({delta}k(t)) observed in single-enzyme experiments were found in a particular study to have an autocorrelation function decaying on the same time scale as that of spectral diffusion {delta}{omega}0(t). To interpret this similarity, the present analysis focuses on a factor in enzyme catalysis, the local electrostatic interaction energy (E) at the active site and its effect on the activation free energy barrier. We consider the slow fluctuations of the electrostatic interaction energy ({delta}E(t)) as a contributor to {delta}k(t) and relate the latter to {delta}{omega}0(t). The resulting relation between {delta}k(t) and {delta}{omega}0(t) is a dynamic analog of the solvatochromism used in interpreting solvent effects on organic reaction rates. The effect of the postulated {delta}E(t) on fluctuations in the radiative component ({delta}{gamma}Formula(t)) of the fluorescence decay of chromophores in proteins also is examined, and a relation between {delta}{gamma}Formula(t) and {delta}{omega}0(t) is obtained. Experimental tests will determine whether the correlation functions for {delta}k(t), {delta}{omega}0(t), and {delta}{gamma}Formula are indeed similar for any enzyme. Measurements of dielectric dispersion, {varepsilon}({omega}), for the enzyme discussed elsewhere will provide further insight into the correlation function for {delta}E(t). They also will determine whether fluctuations in the nonradiative component {gamma}Formula of the lifetime decay has a different origin, fluctuations in distance for example

PH-responsive polysaccharide-based polyelectrolyte complexes as nanocarriers for lysosomal delivery of therapeutic proteins

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Editorial: ImmunoPhysics and ImmunoEngineering

© 2020 Bernardino de la Serna, Mellado, Dustin, Garcia-Parajo and Morikis.The immune system comprises a collection of specialized cells, tissues, and organs that protect the organisms against pathogens and can survey cancer cells. Immune responses are precisely coordinated events that take place in complex, specialized tissue microenvironments. For an integrated view of innate and adaptive immune responses at the molecular level, we ideally need a better understanding of how immune cells communicate and fulfill their tasks in vivo, following events spatially and temporally. Conventional biochemical and genetic methods consider the cell as an individual entity and ligand/receptor pairs as isolated systems. Often, the data obtained refers to the average behavior of a pool of cells and/or receptors removed from their real-life context. The use of new technologies, particularly real-time imaging approaches, is showing us that biological responses are very dynamic and extremely dependent on the context in which they take place and are therefore much more diverse than we initially thought. The combination of these new approaches is radically transforming and enriching immunology, as demonstrated by the increasing number of publications in which physical and/or engineering tools are applied to study the immune response. Whilst scientists are often questioned for the discipline their research is best framed in, we rather think that one scientific discipline cannot be reduced to the terms of another. However, defining and naming cross-disciplinary fields sets our minds on common ground and helps establish a fluent communication to eventually produce groundbreaking, beautiful pieces of science. For instance, ImmunoPhysics was probably first coined by Prof. Morikis a couple of decades ago (https://www.biophysics.org/profiles/dimitrios-morikis); nevertheless, ImmunoPhysics has not become widely regarded as a discipline, despite the continuously growing body of research that requires physical approaches to resolving immunological questions. Hence, with this special issue, we wanted to open a scientific platform compiling ImmunoPhysics and ImmunoEngineering research breakthroughs and future perspectives. Sadly, towards the end of this fascinating journey Prof. Morikis passed away; thus now, with this special issue, we would also like to pay tribute to his fundamental contributions to the field

Planar Optical Nanoantennas Resolve Cholesterol-Dependent Nanoscale Heterogeneities in the Plasma Membrane of Living Cells

Optical nanoantennas can efficiently confine light into nanoscopic hotspots, enabling single-molecule detection sensitivity at biological relevant conditions. This innovative approach to breach the diffraction limit offers a versatile platform to investigate the dynamics of individual biomolecules in living cell membranes and their partitioning into cholesterol-dependent lipid nanodomains. Here, we present optical nanoantenna arrays with accessible surface hotspots to study the characteristic diffusion dynamics of phosphoethanolamine (PE) and sphingomyelin (SM) in the plasma membrane of living cells at the nanoscale. Fluorescence burst analysis and fluorescence correlation spectroscopy performed on nanoantennas of different gap sizes show that, unlike PE, SM is transiently trapped in cholesterol-enriched nanodomains of 10 nm diameter with short characteristic times around 100 μs. The removal of cholesterol led to the free diffusion of SM, consistent with the dispersion of nanodomains. Our results are consistent with the existence of highly transient and fluctuating nanoscale assemblies enriched by cholesterol and sphingolipids in living cell membranes, also known as lipid rafts. Quantitative data on sphingolipids partitioning into lipid rafts is crucial to understand the spatiotemporal heterogeneous organization of transient molecular complexes on the membrane of living cells at the nanoscale. The proposed technique is fully biocompatible and thus provides various opportunities for biophysics and live cell research to reveal details that remain hidden in confocal diffraction-limited measurements.Peer ReviewedPostprint (author's final draft

Moulded photoplastic probes for near-field optical applications

The inexpensive fabrication of high-quality probes for nearfield optical applications is still unsolved although several methods for integrated fabrication have been proposed in the past. A further drawback is the intensity loss of the transmitted light in the 'cut-off' region near the aperture in tapered optical fibres typically used as near-field probes. As a remedy for these limitations we suggest here a new waferscale semibatch microfabrication process for transparent photoplastic probes. The process starts with the fabrication of a pyramidal mould in silicon by using the anisotropic etchant potassium hydroxide. This results in an inverted pyramid limited by silicon crystal planes having an angle of ~54°. The surface including the mould is covered by a ,1.5 nm thick organic monolayer of dodecyltrichlorosilane (DTS) and a 100-nm thick evaporated aluminium film. Two layers of photoplastic material are then spin-coated (thereby conformal filling the mould) and structured by lithography to form a cup for the optical fibre microassembly. The photoplastic probes are finally lifted off mechanically from the mould with the aluminium coating. Focused ion beam milling has been used to subsequently form apertures with diameters in the order of 80 nm. The advantage of our method is that the light to the aperture area can be directly coupled into the probe by using existing fibre-based NSOM set-ups, without the need for far-field alignment, which is typically necessary for cantilevered probes. We have evidence that the aluminium layer is considerably smoother compared to the 'grainy' layers typically evaporated on free-standing probes. The optical throughput efficiency was measured to be about 10^-4. This new NSOM probe was directly bonded to a tuning fork sensor for the shear force control and the topography of a polymer sample was successfully obtained

Priming by Chemokines Restricts Lateral Mobility of the Adhesion Receptor LFA-1 and Restores Adhesion to ICAM-1 Nano-Aggregates on Human Mature Dendritic Cells

LFA-1 is a leukocyte specific β2 integrin that plays a major role in regulating adhesion and migration of different immune cells. Recent data suggest that LFA-1 on mature dendritic cells (mDCs) may function as a chemokine-inducible anchor during homing of DCs through the afferent lymphatics into the lymph nodes, by transiently switching its molecular conformational state. However, the role of LFA-1 mobility in this process is not yet known, despite that the importance of lateral organization and dynamics for LFA-1-mediated adhesion regulation is broadly recognized. Using single particle tracking approaches we here show that LFA-1 exhibits higher mobility on resting mDCs compared to monocytes. Lymphoid chemokine CCL21 stimulation of the LFA-1 high affinity state on mDCs, led to a significant reduction of mobility and an increase on the fraction of stationary receptors, consistent with re-activation of the receptor. Addition of soluble monomeric ICAM-1 in the presence of CCL21 did not alter the diffusion profile of LFA-1 while soluble ICAM-1 nano-aggregates in the presence of CCL21 further reduced LFA-1 mobility and readily bound to the receptor. Overall, our results emphasize the importance of LFA-1 lateral mobility across the membrane on the regulation of integrin activation and its function as adhesion receptor. Importantly, our data show that chemokines alone are not sufficient to trigger the high affinity state of the integrin based on the strict definition that affinity refers to the adhesion capacity of a single receptor to its ligand in solution. Instead our data indicate that nanoclustering of the receptor, induced by multi-ligand binding, is required to maintain stable cell adhesion once LFA-1 high affinity state is transiently triggered by inside-out signals.Peer ReviewedPostprint (published version

A progesterone derivative linked to a stable phospholipid activates breast cancer cell response without leaving the cell membrane

In hormone-responsive breast cancer cells, progesterone (P4) has been shown to act via its nuclear receptor (nPR), a ligand-activated transcription factor. A small fraction of progesterone receptor is palmitoylated and anchored to the cell membrane (mbPR) forming a complex with estrogen receptor alpha (ERα). Upon hormone exposure, either directly or via interaction with ERα, mbPR activates the SRC/RAS/ERK kinase pathway leading to phosphorylation of nPR by ERK. Kinase activation is essential for P4 gene regulation, as the ERK and MSK1 kinases are recruited by the nPR to its genomic binding sites and trigger chromatin remodeling. An interesting open question is whether activation of mbPR can result in gene regulation in the absence of ligand binding to intracellular progesterone receptor (iPR). This matter has been investigated in the past using P4 attached to serum albumin, but the attachment is leaky and albumin can be endocytosed and degraded, liberating P4. Here, we propose a more stringent approach to address this issue by ensuring attachment of P4 to the cell membrane via covalent binding to a stable phospholipid. This strategy identifies the actions of P4 independent from hormone binding to iPR. We found that a membrane-attached progestin can activate mbPR, the ERK signaling pathway leading to iPR phosphorylation, initial gene regulation and entry into the cell cycle, in the absence of detectable intracellular progestin.Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. This work was supported in part by the Spanish Ministerio de Ciencia e Innovación (MCI), Agencia Estatal de Investigación (AEI) and Fondo Europeo de Desarrollo Regional (FEDER) (grant No. PGC2018-099857-B-I00), by the Basque Government (grants No. IT1625-22 and IT1270-19), by Fundación Ramón Areces (CIVP20A6619), by Fundación Biofísica Bizkaia, and by the Basque Excellence Research Centre (BERC) program of the Basque Government. This research was also supported by European Research Council (Project "4D Genome" 609989), the Ministerio de Economía y Competitividad (Project G62426937) and the Generalitat de Catalunya (Project AGAUR SGR 575 and AGAUR 2019PROD00115/IU68-016733), European Research Council -Proof Of Concept (Project “Impacct” 825176). This work has benefited from the equipment and framework of the COMP-HUB and COMP-R Initiatives, funded by the 'Departments of Excellence' Program of the Italian Ministry for University and Research (MIUR, 2018–2022 and MUR, 2022–2027).Peer reviewe

Membrane nanoclusters of FcγRI segregate from inhibitory SIRPα upon activation of human macrophages

Signal integration between activating Fc receptors and inhibitory signal regulatory protein α (SIRPα) controls macrophage phagocytosis. Here, using dual-color direct stochastic optical reconstruction microscopy, we report that Fcγ receptor I (FcγRI), FcγRII, and SIRPα are not homogeneously distributed at macrophage surfaces but are organized in discrete nanoclusters, with a mean radius of 71 ± 11 nm, 60 ± 6 nm, and 48 ± 3 nm, respectively. Nanoclusters of FcγRI, but not FcγRII, are constitutively associated with nanoclusters of SIRPα, within 62 ± 5 nm, mediated by the actin cytoskeleton. Upon Fc receptor activation, Src-family kinase signaling leads to segregation of FcγRI and SIRPα nanoclusters to be 197 ± 3 nm apart. Co-ligation of SIRPα with CD47 abrogates nanocluster segregation. If the balance of signals favors activation, FcγRI nanoclusters reorganize into periodically spaced concentric rings. Thus, a nanometer- and micron-scale reorganization of activating and inhibitory receptors occurs at the surface of human macrophages concurrent with signal integration