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In neurons, endoplasmic reticulum forms a highly dynamic network that enters axons and presynaptic terminals and plays a central role in Ca2+ homeostasis and synapse maintenance. However, the underlying mechanisms involved in regulation of its dynamic remodeling as well as its function in axon development and presynaptic differentiation remain elusive. Here, we used high resolution microscopy and live cell imaging to investigate rapid movements of endoplasmic reticulum and ribosomes in axons of cultured motoneurons after stimulation with Brain-derived neurotrophic factor. Our results indicate that the endoplasmic reticulum extends into axonal growth cone filopodia where its integrity and dynamic remodeling are regulated mainly by actin and its motor protein myosin VI. Additionally, we found that in axonal growth cones, ribosomes assemble into 80S subunits within seconds and associate with ER in response to extracellular stimuli which describes a novel function of axonal ER in dynamic regulation of local translation.Summary statementIn axonal growth cones of motoneurons, dynamic remodeling of ER is coordinated through drebrin A-mediated actin and microtubule crosstalk and contributes to local translation as response to BDNF stimulation.

We have developed a new functionalization approach for semiconductor nanocrystals based on a single-step exchange of surface ligands with custom-designed peptides. This peptide-coating technique yield small, monodisperse and very stable water-soluble NCs that remain bright and photostable. We have used this approach on several types of core and core-shell NCs in the visible and near-infrared spectrum range and used fluorescence correlation spectroscopy for rapid assessment of the colloidal and photophysical properties of the resulting particles. This peptide coating strategy has several advantages: it yields probes that are immediately biocompatible; it is amenable to improvements of the different properties (solubilization, functionalization, etc) via rational design, parallel synthesis, or molecular evolution; it permits the combination of several functions on individual NCs. These functionalized NCs have been used for diverse biomedical applications. Two are discussed here: single-particle tracking of membrane receptor in live cells and combined fluorescence and PET imaging of targeted delivery in live animals.

Besides its function as a passive cell wall, the plasma membrane (PM) serves as a platform for different physiological processes such as signal transduction and cell adhesion, determining the ability of cells to communicate with the exterior, and form tissues. Therefore, the spatial distribution of PM components, and the molecular mechanisms underlying it, have important implications in various biological fields including cell development, neurobiology, and immunology. The existence of confined compartments in the plasma membrane that vary on many length scales from protein multimers to micrometer-size domains with different protein and lipid composition is today beyond all questions. As much as the physiology of cells is controlled by the spatial organization of PM components, the study of distribution, size, and composition remains challenging. Visualization of the molecular distribution of PM components has been impeded mainly due to two problems: the specific labeling of lipids and proteins without perturbing their native distribution and the diffraction-limit of fluorescence microscopy restricting the resolution to about half the wavelength of light. Here, we present a bioorthogonal chemical reporter strategy based on click chemistry and metabolic labeling for efficient and specific visualization of PM proteins and glycans with organic fluorophores in combination with super-resolution fluorescence imaging by direct stochastic optical reconstruction microscopy (dSTORM) with single-molecule sensitivity.

▪ Abstract Recent years have witnessed a renaissance of fluorescence microscopy techniques and applications, from live-animal multiphoton confocal microscopy to single-molecule fluorescence spectroscopy and imaging in living cells. These achievements have been made possible not so much because of improvements in microscope design, but rather because of development of new detectors, accessible continuous wave and pulsed laser sources, sophisticated multiparameter analysis on one hand, and the development of new probes and labeling chemistries on the other. This review tracks the lineage of ideas and the evolution of thinking that have led to the actual developments, and presents a comprehensive overview of the field, with emphasis put on our laboratory's interest in single-molecule microscopy and spectroscopy.

Stacking interactions between organic fluorophores can cause formation of non-fluorescent H-dimers. Dimer formation and dissociation of two fluorophores site-specifically incorporated in a biomolecule result in fluorescence intermittency that can report on conformational dynamics. We characterize intramolecular dimerization of two oxazine fluorophores MR121 attached to an unstructured polypeptide. Formation of stable non-fluorescent complexes with nano- to microsecond lifetimes is a prerequisite for analysing the intermittent fluorescence emission by fluorescence correlation spectroscopy and extracting relaxation time constants on nano- to millisecond time scales. Destabilization of the generally very stable homodimers by chemical denaturation reduces the lifetime of H-dimers. We demonstrate that H-dimer formation of an oxazine fluorophore reports on end-to-end contact rates in unstructured glycine-serine polypeptides under denaturing conditions.

Characterization of the length dependence of end-to-end loop-closure kinetics in unfolded polypeptide chains provides an understanding of early steps in protein folding. Here, loop-closure in poly-glycine-serine peptides is investigated by combining single-molecule fluorescence spectroscopy with molecular dynamics simulation. For chains containing more than 10 peptide bonds loop-closing rate constants on the 20–100 nanosecond time range exhibit a power-law length dependence. However, this scaling breaks down for shorter peptides, which exhibit slower kinetics arising from a perturbation induced by the dye reporter system used in the experimental setup. The loop-closure kinetics in the longer peptides is found to be determined by the formation of intra-peptide hydrogen bonds and transient β-sheet structure, that accelerate the search for contacts among residues distant in sequence relative to the case of a polypeptide chain in which hydrogen bonds cannot form. Hydrogen-bond-driven polypeptide-chain collapse in unfolded peptides under physiological conditions found here is not only consistent with hierarchical models of protein folding, that highlights the importance of secondary structure formation early in the folding process, but is also shown to speed up the search for productive folding events., Author Summary In studies of protein folding evidence exists for early compaction in the unfolded state, although it is unclear whether these compact conformations contain specific secondary structures (through hydrophilic interactions) or whether compaction is a non-specific hydrophobic-driven effect. Here we combine single-molecule fluorescence spectroscopy and molecular dynamics simulation to demonstrate peptide hydrogen-bond-driven polypeptide-chain collapse involving secondary structure formation as the key process in the early stage of folding. Partial structuring in unfolded polypeptide chains is shown to lead to faster contact formation kinetics than would be expected if the unfolded state were populated by featureless random-coils.

Fluorescence correlation spectroscopy is applied on homologous human lectins (i.e., adhesion/growth-regulatory galectins) to detect influence of ligand binding and presence of the linker peptide in tandem-repeat-type proteins on hydrodynamic properties. Among five tested proteins, lactose binding increased the diffusion constant only in the cases of homodimeric galectin-1 and the linkerless variant of tandem-repeat-type galectin-4. To our knowledge, the close structural similarity among galectins does not translate into identical response to ligand binding. Kinetic measurements show association and dissociation rate constants in the order of 1 to 10(3) M(-1) s(-1) and 10(-4) s(-1), respectively. Presence of the linker peptide in tandem-repeat-type protein leads to anomalous scaling with molecular mass. These results provide what we believe to be new insights into lectin responses to glycan binding, detectable so far only by small angle neutron scattering, and the structural relevance of the linker peptide. Methodologically, fluorescence correlation spectroscopy is shown to be a rather simple technical tool to characterize hydrodynamic properties of these proteins at a high level of sensitivity.

Polyprolines are well known for adopting a regular polyproline type II helix in aqueous solution, rendering them a popular standard as molecular ruler in structural molecular biology. However, single-molecule spectroscopy studies based on Förster resonance energy transfer (FRET) have revealed deviations of experimentally observed end-to-end distances of polyprolines from theoretical predictions, and it was proposed that the discrepancy resulted from dynamic flexibility of the polyproline helix. Here, we probe end-to-end distances and conformational dynamics of poly- l -prolines with 1–10 residues using fluorescence quenching by photoinduced-electron transfer (PET). A single fluorophore and a tryptophan residue, introduced at the termini of polyproline peptides, serve as sensitive probes for distance changes on the subnanometer length scale. Using a combination of ensemble fluorescence and fluorescence correlation spectroscopy, we demonstrate that polyproline samples exhibit static structural heterogeneity with subpopulations of distinct end-to-end distances that do not interconvert on time scales from nano- to milliseconds. By observing prolyl isomerization through changes in PET quenching interactions, we provide experimental evidence that the observed heterogeneity can be explained by interspersed cis isomers. Computer simulations elucidate the influence of trans/cis isomerization on polyproline structures in terms of end-to-end distance and provide a structural justification for the experimentally observed effects. Our results demonstrate that structural heterogeneity inherent in polyprolines, which to date are commonly applied as a molecular ruler, disqualifies them as appropriate tool for an accurate determination of absolute distances at a molecular scale.

In recent years Fluorescence Resonance Energy Transfer (FRET) has been widely used to determine distances, observe distance dynamics, and monitor molecular binding at the single-molecule level. A basic constraint of single-molecule FRET studies is the limited distance resolution owing to low photon statistics. We demonstrate that by confining molecules in nanopipettes (50-100 nm diameter) smFRET can be measured with improved photon statistics reducing the width of FRET proximity ratio distributions (PRD). This increase in distance resolution makes it possible to reveal subpopulations and dynamics in biomolecular complexes. Our data indicate that the width of PRD is not only determined by photon statistics (shot noise) and distance distributions between the chromophores but that photoinduced dark states of the acceptor also contribute to the PRD width. Furthermore, acceptor dark states such as triplet states influence the accuracy of determined mean FRET values. In this context, we present a strategy for the correction of the shift of the mean PR that is related to triplet induced blinking of the acceptor using reference FCS measurements.

Colloidal NCs consist of an inorganic particle and an organic coating that determines their solubility, functionality, and influences their photophysics. In order for these NCs to be biocompatible, they must be water-soluble, nontoxic to the cell, and offer conjugation chemistries for attaching recognition molecules to their surfaces. In addition they should efficiently target to biomolecules of interest, be chemically stable, and preserve their high photostability. The requirements for their application in single-molecule biological studies are even more stringent: fluorescent NCs should be monodisperse, have relatively small size (to limit steric hindrance), reduced blinking, large saturation intensity, and high quantum yield (QY).