Human α-Synuclein (αSyn) is a natively unfolded protein whose aggregation into amyloid fibrils is involved in the pathology of Parkinson disease. A full comprehension of the structure and dynamics of early intermediates leading to the aggregated states is an unsolved problem of essential importance to researchers attempting to decipher the molecular mechanisms of αSyn aggregation and formation of fibrils. Traditional bulk techniques used so far to solve this problem point to a direct correlation between αSyn's unique conformational properties and its propensity to aggregate, but these techniques can only provide ensemble-averaged information for monomers and oligomers alike. They therefore cannot characterize the full complexity of the conformational equilibria that trigger the aggregation process. We applied atomic force microscopy–based single-molecule mechanical unfolding methodology to study the conformational equilibrium of human wild-type and mutant αSyn. The conformational heterogeneity of monomeric αSyn was characterized at the single-molecule level. Three main classes of conformations, including disordered and “β-like” structures, were directly observed and quantified without any interference from oligomeric soluble forms. The relative abundance of the “β-like” structures significantly increased in different conditions promoting the aggregation of αSyn: the presence of Cu2+, the pathogenic A30P mutation, and high ionic strength. This methodology can explore the full conformational space of a protein at the single-molecule level, detecting even poorly populated conformers and measuring their distribution in a variety of biologically important conditions. To the best of our knowledge, we present for the first time evidence of a conformational equilibrium that controls the population of a specific class of monomeric αSyn conformers, positively correlated with conditions known to promote the formation of aggregates. A new tool is thus made available to test directly the influence of mutations and pharmacological strategies on the conformational equilibrium of monomeric αSyn.

Unfixed chicken erythrocyte chromatin fibers in very low salt have been imaged with a nning force microscope operating in the tapping mode in air at ambient humidity. These images reveal a threedimensional organizaffon of the fibers. The planar "6zig-zag" conformation is rare, and extended "beads-on-a-string" fibers are seen only in chromatin depleted of histes Hi and H5. Glutaraldehyde fixation reveals very similar structures. Fibers fixed in 10 mM salt appear somewhat more compacted. These results, when compared with modeing stude, suggest that chromatin fibers may exist as frregular three-dimensional arrays of nucleosomes even at low ionic strength.The structure of the chromatin fiber in low salt concentrations remains controversial. Electron microscopy (EM) experiments, most of which utilized the Miller spreading technique (1), typically showed extended "beads-on-a-string" or "open zig-zag" structures (refs. 2 and 3; for reviews, see refs. 4-6). At slightly higher ionic strength (-10 mM NaCI), somewhat more compact, "closed" zig-zags of nucleosomes were observed (7-9). Only upon further addition of NaCl to about 100 mM did these extended structures condense to form the so-called 30-nm fiber (8), which resembles structures observed in situ (7, 9, 10). However, there has been concern that strong interactions of the fiber with the EM support surface, and the dehydration produced by the high vacuum conditions, could distort the structure, especially at low ionic strength.Attempts to circumvent these problems used solution studies. The first scattering experiments suggested that the nucleosomes were densely packed in a linear array (11,12 To address this controversy, a study was performed using the three-dimensional imagi capabilities ofa scanning force microscope, which makes it possible to image chromatin fibers under less damaging conditions (22,23). The samples are never vacuum dried and are scanned in air at about 50%o relative humidity. Under these conditions a film of liquid water resides on the support surface (24). The newly developed tapping operation mode (25, 26) was employed, in which a stiff cantilever is oscillated near its resonance frequency with amplitudes typically in the range of 10-20 nm as the sample is scanned laterally. The oscillation amplitude is kept constant via feedback control. This operation has several advantages over the contact mode, in which a tip is pulled across the sample. Tip-sample forces are lighter than in the contact mode. Moreover, since most of the force is perpendicular to the surface, the sample experiences minimal lateral deformation during scanning, thus improving spatial resolution (25, 26). MATERIAL AND METHODSPreparation and Fization of Chromatin. Chicken erythrocyte chromatin was prepared essentially as described (27), with a reduction in the amount of micrococcal nuclease to allow isolation of long fibers (28). Soluble chromatin was dialyzed versus 5 mM triethanolamine/HC1 (pH 7.0), with or without 10 mM NaCl and was stored on ice. In a few experiments...

No abstract

The molecular mechanisms that regulate coordinated and colinear activation of Hox gene expression in space and time remain poorly understood. Here we demonstrate that Plzf regulates the spatial expression of the AbdB HoxD gene complex by binding to regulatory elements required for restricted Hox gene expression and can recruit histone deacetylases to these sites. We show by scanning forced microscopy that Plzf, via homodimerization, can form DNA loops and bridge distant Plzf binding sites located within HoxD gene regulatory elements. Furthermore, we demonstrate that Plzf physically interacts with Polycomb proteins on DNA. We propose a model by which the balance between activating morphogenic signals and transcriptional repressors such as Plzf establishes proper Hox gene expression boundaries in the limb bud.

No abstract