Far-red cyanine fluorophores find extensive use in modern microscopy despite modest quantum yields. To improve the photon output of these molecules, we report a synthetic strategy that blocks the major deactivation pathway: excited-state trans-to-cis polyene rotation. In the key transformation, a protected dialdehyde precursor undergoes a cascade reaction to install the requisite tetracyclic ring system. The resulting molecules exhibit the characteristic features of conformational restraint, including improved fluorescence quantum yield and extended lifetime. Moreover, these compounds recover from hydride reduction with dramatically improved efficiency. These observations enable efficient single-molecule localization microscopy in oxygenated buffer without addition of thiols. Enabled by modern organic synthesis, these studies provide a new class of far-red dyes with promising spectroscopic and chemical properties.
Synaptonemal complexes (SCs) are meiosis-specific multiprotein complexes that are essential for synapsis, recombination, and segregation of homologous chromosomes, but the molecular organization of SCs remains unclear. We used immunofluorescence labeling in combination with super-resolution imaging and average position determination to investigate the molecular architecture of SCs. Combination of 2D super-resolution images recorded from different areas of the helical ladder-like structure allowed us to reconstruct the 3D molecular organization of the mammalian SC with isotropic resolution. The central element is composed of two parallel cables at a distance of ∼100 nm, which are oriented perpendicular to two parallel cables of the lateral element arranged at a distance of ∼220 nm. The two parallel cable elements form twisted helical structures that are connected by transversal filaments by their N and C termini. A single-cell preparation generates sufficient localizations to compile a 3D model of the SC with nanometer precision.super-resolution imaging | synaptonemal complex | dSTORM | meiosis | average position determination T he synaptonemal complex (SC) is a well-preserved meiosisspecific protein complex among different species (1, 2). As revealed by transmission electron microscopy (TEM), when fully assembled, SCs are 200-nm-wide, ribbon-like structures that extend all along a chromosome bivalent (3, 4). SCs have a characteristic ladder-like organization that is highly conserved through evolution and consists of two lateral elements (LEs), at which chromatin of homologous chromosomes is attached, and a central region (CR). The CR holds the homologous chromosomes together and is made up of numerous transversal filaments (TFs) and the central element (CE).At present, seven protein components of the synaptonemal complex have been identified in mammals, namely, the LE proteins SYCP2 and SYCP3 (5, 6); the TF protein SYCP1 (7); and the CE-specific proteins SYCE1, SYCE2, SYCE3, and TEX12 (8-10) (Figs. S1 and S2). However, in addition to the identification of SC protein components and the investigation of interaction partners, the establishment of a model of the molecular architecture remains indispensable for the understanding of its function and assembly process. Due to the resolution limit of conventional fluorescence microscopy, information about the molecular organization of SCs has been mainly obtained by immunogold EM (11). In standard immunogold EM preparations, gold particles are localized with nanometer resolution and the localization precision is mainly limited by the size of the primary and secondary IgG antibodies and the gold signal density (12-14). However, sample preparation is time-consuming and quantitative analysis of the signal is tedious because of the low binding efficiency of goldlabeled antibodies. Because the structural resolution is also determined by the labeling density (15), immunogold EM cannot visualize the different SC proteins as continuous structures. Therefore, the construction of ...
In outside-out patches of mouse-muscle membrane, embryonic-like channels were activated by pulses of acetylcholine (ACh). On increasing the ACh concentration, the rate of desensitization, 1/tau d, increased linearly with the peak open probability, indicating desensitization from the open state. Desensitization had only one time constant tau d at each ACh concentration. Recovery from desensitization was only approximately 10 times slower than desensitization, whereas the probability of steady-state channel opening, declined to < 0.01 with > 10(-6) M ACh. The peak probability of opening in > 10(-4) M ACh pulse was close to 1. A linear reaction scheme was not compatible with these results. The scheme had to be expanded resulting in a circular scheme with two additional ACh binding steps to desensitized channel states. The approximate rate constants of all reaction steps in the circular scheme could be determined using computer simulations. The model predicted that clusters of channel opening had the average duration tau d at the respective ACh concentration. In cell-attached patches on intact muscle fibers, similar average cluster durations were observed at the respective ACh concentration. This indicates that tau d in the intact muscle fibers has similar values as in outside-out patches.
We studied the kinetics of the unedited version of rat GluR6 glutamate (glu) receptor channels, GluR6Q, in outside-out patches using a system for submillisecond solution exchange. Half-maximum activation of the channels was reached with approximately 0.5 microM glu. The maximum slope of the double-logarithmic plot of the peak current versus glu was approximately 1.3, indicating that at least two binding steps are necessary to open the channels. Currents in response to a pulse of 10 microM glu had a short rise time (10-90% of peak current) of approximately 220 microseconds at approximately 20 degrees C. The rise time increased with falling glu concentration, reaching approximately 6.0 ms with 10 microM glu. In the continued presence of glu, the channels desensitized, and this desensitization can be described with a single time constant of approximately 7.0 ms for a pulse of 10 microM glu. The steady-state current in response to a long pulse of 10 microM glu was below 1/280th of the peak current. The time constant of desensitization was found to be independent of concentration between 30.0 and 0.3 microM glu, but to be increased for lower concentrations. After a short pulse of 1 ms duration and 10 or 0.3 microM glu, currents decayed with a time constant of approximately 2.5 ms. Recovery from desensitization after a pulse took approximately 5 s, and the half-time of recovery was approximately 2.2 s. Continuous application of low concentrations of glutamate reduced the peak currents in response to a pulse of 10 microM glu markedly. Fifty percent response reduction was observed in the continuous presence of approximately 0.3 microM glu. Our results for homomeric GluR6 agree with a cyclical reaction scheme developed for completely desensitizing, glu-activated channels on crayfish muscles.
Here, we combine super-resolution fluorescence localization microscopy with scanning electron microscopy to map the position of proteins of nuclear pore complexes in isolated Xenopus laevis oocyte nuclear envelopes with molecular resolution in both imaging modes. We use the periodic molecular structure of the nuclear pore complex to superimpose direct stochastic optical reconstruction microscopy images with a precision of ,20 nm on electron micrographs. The correlative images demonstrate quantitative molecular labeling and localization of nuclear pore complex proteins by standard immunocytochemistry with primary and secondary antibodies and reveal that the nuclear pore complex is composed of eight gp210 (also known as NUP210) protein homodimers. In addition, we find subpopulations of nuclear pore complexes with ninefold symmetry, which are found occasionally among the more typical eightfold symmetrical structures.
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