Thank you! Your submission has been received!
Oops! Something went wrong while submitting the form.
The β-barrel assembly machinery (BAM) complex is responsible for inserting outer membrane proteins (OMPs) into the Escherichia coli outer membrane. The SecYEG translocon inserts inner membrane proteins into the inner membrane and translocates both soluble proteins and nascent OMPs into the periplasm. Recent reports describe Sec possibly playing a direct role in OMP biogenesis through interactions with the soluble polypeptide transport-associated (POTRA) domains of BamA (the central OMP component of BAM). Here we probe the diffusion behavior of these protein complexes using photoactivatable super-resolution localization microscopy and single-particle tracking in live E. coli cells of BAM and SecYEG components BamA and SecE and compare them to other outer and inner membrane proteins. To accurately measure trajectories on the highly curved cell surface, three-dimensional tracking was performed using double-helix point-spread function microscopy. All proteins tested exhibit two diffusive modes characterized by “slow” and “fast” diffusion coefficients. We implement a diffusion coefficient analysis as a function of the measurement lag time to separate positional uncertainty from true mobility. The resulting true diffusion coefficients of the slow and fast modes showed a complete immobility of full-length BamA constructs in the time frame of the experiment, whereas the OMP OmpLA displayed a slow diffusion consistent with the high viscosity of the outer membrane. The periplasmic POTRA domains of BamA were found to anchor BAM to other cellular structures and render it immobile. However, deletion of individual distal POTRA domains resulted in increased mobility, suggesting that these domains are required for the full set of cellular interactions. SecE diffusion was much slower than that of the inner membrane protein PgpB and was more like OMPs and BamA. Strikingly, SecE diffused faster upon POTRA domain deletion. These results are consistent with the existence of a BAM-SecYEG trans-periplasmic assembly in live E. coli cells.
To understand how the nucleosome remodeling and deacetylase (NuRD) complex regulates enhancers and enhancer–promoter interactions, we have developed an approach to segment and extract key biophysical parameters from live-cell three-dimensional single-molecule trajectories. Unexpectedly, this has revealed that NuRD binds to chromatin for minutes, decompacts chromatin structure and increases enhancer dynamics. We also uncovered a rare fast-diffusing state of enhancers and found that NuRD restricts the time spent in this state. Hi-C and Cut&Run experiments revealed that NuRD modulates enhancer–promoter interactions in active chromatin, allowing them to contact each other over longer distances. Furthermore, NuRD leads to a marked redistribution of CTCF and, in particular, cohesin. We propose that NuRD promotes a decondensed chromatin environment, where enhancers and promoters can contact each other over longer distances, and where the resetting of enhancer–promoter interactions brought about by the fast decondensed chromatin motions is reduced, leading to more stable, long-lived enhancer–promoter relationships.
Proteins critical for synaptic transmission are non-uniformly distributed and assembled into regions of high density called subsynaptic densities (SSDs) that transsynaptically align in nanocolumns. Neurexin-1 and neurexin-3 are essential presynaptic adhesion molecules that non-redundantly control NMDAR- and AMPAR-mediated synaptic transmission, respectively, via transsynaptic interactions with distinct postsynaptic ligands. Despite their functional relevance, fundamental questions regarding the nanoscale properties of individual neurexins, their influence on the subsynaptic organization of excitatory synapses and the mechanisms controlling how individual neurexins engage in precise transsynaptic interactions are unknown. Using Double Helix 3D dSTORM and neurexin mouse models, we identify neurexin-3 as a critical presynaptic adhesion molecule that regulates excitatory synapse nano-organization in hippocampus. Furthermore, endogenous neurexin-1 and neurexin-3 form discrete and non-overlapping SSDs that are enriched opposite their postsynaptic ligands. Thus, the nanoscale organization of neurexin-1 and neurexin-3 may explain how individual neurexins signal in parallel to govern different synaptic properties.
Significance: Double-helix point spread function (DH-PSF) microscopy has been developed for three-dimensional (3D) localization and imaging at super-resolution but usually in environments with no or weak scattering. To date, super-resolution imaging through turbid media has not been reported. Aim: We aim to explore the potential of DH-PSF microscopy in the imaging and localization of targets in scattering environments for improved 3D localization accuracy and imaging quality. Approach: The conventional DH-PSF method was modified to accommodate the scanning strategy combined with a deconvolution algorithm. The localization of a fluorescent microsphere is determined by the center of the corresponding double spot, and the image is reconstructed from the scanned data by deconvoluting the DH-PSF. Results: The resolution, i.e., the localization accuracy, was calibrated to 13 nm in the transverse plane and 51 nm in the axial direction. Penetration thickness could reach an optical thickness (OT) of 5. Proof-of-concept imaging and the 3D localization of fluorescent microspheres through an eggshell membrane and an inner epidermal membrane of an onion are presented to demonstrate the super-resolution and optical sectioning capabilities. Conclusions: Modified DH-PSF microscopy can image and localize targets buried in scattering media using super-resolution. Combining fluorescent dyes, nanoparticles, and quantum dots, among other fluorescent probes, the proposed method may provide a simple solution for visualizing deeper and clearer in/through scattering media, making in situ super-resolution microscopy possible for various demanding applications.
We developed a multi-unit microscope for all-optical inter-layers circuits interrogation. The system performs two-photon (2P) functional imaging and 2P multiplexed holographic optogenetics at axially distinct planes. We demonstrated the capability of the system to map, in the mouse retina, the functional connectivity between rod bipolar cells (RBCs) and ganglion cells (GCs) by activating single or defined groups of RBCs while recording the evoked response in the GC layer with cell-type specificity and single-cell resolution. We then used a logistic model to probe the functional connectivity between cell types by deriving the ‘‘cellular receptive field’’ describing how RBCs impact each GC type. With the capability to simultaneously image and control neuronal activity at axially distinct planes, the system enables a precise interrogation of multi-layered circuits. Understanding this information transfer is a promising avenue to dissect complex neural circuits and understand the neural basis of computations.
Mitochondria are essential organelles of eukaryotic cells with key functions in metabolism, apoptosis, and signaling. As a result, impaired mitochondrial function has been associated with numerous diseases. In order to understand mitochondrial processes, it is fundamental to gain knowledge about their structure and microcompartmentalization, including the function, organization, and dynamics of their protein, nucleic acid, and lipid components. A number of recent groundbreaking advances in fluorescence microscopy enable the study of mitochondrial biology with unprecedented detail. Among them, new methods based on single-molecule and super-resolution microscopy allow us to study mitochondrial structures, protein organizations, and dynamics. Here, we discuss the advantages and disadvantages of different single-molecule microscopy methods to study individual proteins in fixed and living cells in the background of mitochondrial processes, in situ.
To understand how cells work, we need elucidate how proteins interact inside cellular ultrastructure. Super-resolution microscopy, e.g. stochastic optical reconstruction microscopy (STORM) , underpins our understanding of interacting molecular networks in cells at the resolution of dozens of nanometres. However, to ascertain protein structure and function relationship, cryogenic correlative light and electron microscopy (cryo-CLEM)  is highly sought after because it combines the functional information from molecular tagging in light microscopy with the intact ultrastructure information in electron microscopy. The challenge is the discrepancy in resolving power and imaging volume between cryo-EM and conventional cryoFM. To address this challenge, we developed cryogenic STORM (cryo-STORM) to achieve sub-10 nm localization precision , and 3D Double Helix STORM with extended imaging volume to a few microns in a single shot. We are developing superresolution cryo-CLEM workflow, aiming at unravelling the structure-function relationship of proteins and their partners throughout the cells with unprecedented precision.
A new super-resolution technique for localisation microscopy, which combines active control of probe photophysics with stochastic binding is reported. resPAINT yields an up to 50-fold improvement in localisation rate vs. PAINT without compromising contrast and is fully compatible with large depth of field imaging techniques. This opens the door to larger scale 3D localisation microscopy as imaging that normally takes days can now be completed in hours.
Chromatin organization and dynamics are critical for gene regulation. In this work we present a methodology for fast and parallel three-dimensional (3D) tracking of multiple chromosomal loci of choice over many thousands of frames on various timescales. We achieved this by developing and combining fluorogenic and replenishable nanobody arrays, engineered point spread functions, and light sheet illumination. The result is gentle live-cell 3D tracking with excellent spatiotemporal resolution throughout the mammalian cell nucleus. Correction for both sample drift and nuclear translation facilitated accurate long-term tracking of the chromatin dynamics. We demonstrate tracking both of fast dynamics (50 Hz) and over timescales extending to several hours, and we find both large heterogeneity between cells and apparent anisotropy in the dynamics in the axial direction. We further quantify the effect of inhibiting actin polymerization on the dynamics and find an overall increase in both the apparent diffusion coefficient D* and anomalous diffusion exponent α and a transition to more-isotropic dynamics in 3D after such treatment. We think that in the future our methodology will allow researchers to obtain a better fundamental understanding of chromatin dynamics and how it is altered during disease progression and after perturbations of cellular function.
No results found
Please try different keywords