Cell Biology


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0 Q&A 322 Views Oct 20, 2023

A fundamental understanding of gene regulation requires a quantitative characterization of the spatial organization and dynamics of chromatin. The advent of fluorescence super-resolution microscopy techniques such as photoactivated localization microscopy (PALM) presented a breakthrough to visualize structural features with a resolution of ~20 nm in fixed cells. However, until recently the long acquisition time of super-resolution images prevented high-resolution measurements in living cells due to spreading of localizations caused by chromatin motion. Here, we present a step-by step protocol for our recently developed approach for correlatively imaging telomeres with conventional fluorescence and PALM, in order to obtain time-averaged super-resolution images and dynamic parameters in living cells. First, individual single molecule localizations are assigned to a locus as it moves, allowing to discriminate between bound and unbound dCas9 molecules, whose mobilities overlap. By subtracting the telomere trajectory from the localization of bound molecules, the motion blurring is then corrected, and high-resolution structural characterizations can be made. These structural parameters can also be related to local chromatin motion or larger scale domain movement. This protocol therefore improves the ability to analyze the mobility and time-averaged nanoscopic structure of locus-specific chromatin with single-molecule sensitivity.

0 Q&A 2001 Views Jun 20, 2023

Expansion microscopy (ExM) is a powerful technique to overcome the diffraction limit of light microscopy that can be applied in both tissues and cells. In ExM, samples are embedded in a swellable polymer gel to physically expand the sample and isotropically increase resolution in x, y, and z. By systematic exploration of the ExM recipe space, we developed a novel ExM method termed Ten-fold Robust Expansion Microscopy (TREx) that, as the original ExM method, requires no specialized equipment or procedures. TREx enables ten-fold expansion of both thick mouse brain tissue sections and cultured human cells, can be handled easily, and enables high-resolution subcellular imaging with a single expansion step. Furthermore, TREx can provide ultrastructural context to subcellular protein localization by combining antibody-stained samples with off-the-shelf small molecule stains for both total protein and membranes.

0 Q&A 348 Views Apr 20, 2023

The mammalian Golgi complex consists of laterally connected Golgi stacks, each comprising close-packed and flattened membrane sacks called cisternae. However, the convoluted spatial organization of Golgi stacks and limited resolution of light microscopy prevent us from resolving the cisternal organization of the Golgi. Here, we describe our recently developed side-averaging approach coupled with Airyscan microscopy to visualize the cisternal organization of nocodazole-induced Golgi ministacks. First, the nocodazole treatment greatly simplifies the organization of Golgi stacks by spatially separating the crowded and amorphous Golgi complex into individual disk-shaped ministacks. The treatment also makes it possible to identify en face and side-views of Golgi ministacks. Next, after manually selecting the side-view Golgi ministack images, they are transformed and aligned. Finally, the resulting images are averaged to enhance the common structural features and suppress the morphological variations among individual Golgi ministacks. This protocol describes how to image and analyze the intra-Golgi localization of giantin, GalT-mCherry, GM130, and GFP-OSBP in HeLa cells by side-averaging.


Graphical overview


0 Q&A 1874 Views Mar 20, 2022

The centrosome is the main microtubule-organizing center of animal cells, and is composed of two barrel-shaped microtubule-based centrioles embedded in protein dense pericentriolar material. Compositional and architectural re-organization of the centrosome drives its duplication, and enables its microtubule-organizing activity and capability to form the primary cilium, which extends from the mature (mother) centriole, as the cell exits the cell cycle. Centrosomes and primary cilia are essential to human health, signified by the causal role of centrosome- and cilia-aberrations in numerous congenic disorders, as well as in the etiology and progression of cancer. The list of disease-associated centrosomal proteins and their proximitomes is steadily expanding, emphasizing the need for high resolution mapping of such proteins to specific substructures of the organelle. Here, we provide a detailed 3D-structured illumination microscopy (3D-SIM) protocol for comparative localization analysis of fluorescently labeled proteins at the centrosome in fixed human cell lines, at approximately 120 nm lateral and 300 nm axial resolution. The procedure was optimized to work with primary antibodies previously known to depend on more disruptive fixation reagents, yet largely preserves centriole and centrosome architecture, as shown by transposing acquired images of landmark proteins on previously published transmission electron microscopy (TEM) images of centrosomes. Even more advantageously, it is compatible with fluorescent protein tags. Finally, we introduce an internal reference to ensure correct 3D channel alignment. This protocol hence enables flexible, swift, and information-rich localization and interdependence analyses of centrosomal proteins, as well as their disorder-associated mutations.

0 Q&A 2782 Views Aug 20, 2021

Analyzing cellular structures and the relative location of molecules is essential for addressing biological questions. Super-resolution microscopy techniques that bypass the light diffraction limit have become increasingly popular to study cellular molecule dynamics in situ. However, the application of super-resolution imaging techniques to detect small RNAs (sRNAs) is limited by the choice of proper fluorophores, autofluorescence of samples, and failure to multiplex. Here, we describe an sRNA-PAINT protocol for the detection of sRNAs at nanometer resolution. The method combines the specificity of locked nucleic acid probes and the low background, precise quantitation, and multiplexable characteristics of DNA Point Accumulation for Imaging in Nanoscale Topography (DNA-PAINT). Using this method, we successfully located sRNA targets that are important for development in maize anthers at sub-20 nm resolution and quantitated their exact copy numbers.


Graphic abstract:



Multiplexed sRNA-PAINT. Multiple Vetting and Analysis of RNA for In Situ Hybridization (VARNISH) probes with different docking strands (i.e., a, b, …) will be hybridized to samples. The first probe will be imaged with the a* imager. The a* imager will be washed off with buffer C, and then the sample will be imaged with b* imager. The wash and image steps can be repeated sequentially for multiplexing.


0 Q&A 2478 Views Nov 5, 2020

Supramolecular signaling assemblies are of interest for their unique signaling properties. A µm scale signaling assembly, the central supramolecular signaling cluster (cSMAC), forms at the center interface of T cells activated by antigen presenting cells (APC). The adaptor protein linker for activation of T cells (LAT) is a key cSMAC component. The cSMAC has widely been studied using total internal reflection fluorescence microscopy of CD4+ T cells activated by planar APC substitutes. Here we provide a protocol to image the cSMAC in its cellular context at the interface between a T cell and an APC. Super resolution stimulated emission depletion microscopy (STED) was utilized to determine the localization of LAT, that of its active, phosphorylated form and its entire pool. Agonist peptide-loaded APCs were incubated with TCR transgenic CD4+ T cells for 4.5 min before fixation and antibody staining. Fixed cell couples were imaged using a 100x 1.4 NA objective on a Leica SP8 AOBS confocal laser scanning microscope. LAT clustered in multiple supramolecular complexes and their number and size distributions were determined. Using this protocol, cSMAC properties in its cellular context at the interface between a T cell and an APC could be quantified.

0 Q&A 4143 Views Sep 5, 2020
Many questions in cell biology can be solved by state-of-the-art technology of live cell imaging. One good example is the mechanism of membrane traffic, in which small membrane carriers are rapidly moving around in the cytoplasm to deliver cargo proteins between organelles. For directly visualizing the events in membrane trafficking system, researchers have long awaited the technology that enables simultaneous multi-color and four-dimensional observation at high space and time resolution. Super-resolution microscopy methods, for example STED, PALM/STORM, and SIM, provide greater spatial resolution, however, these methods are not enough in temporal resolution. The super-resolution confocal live imaging microscopy (SCLIM) that we developed has now achieved the performance required. By using SCLIM, we have conducted high spatiotemporal visualization of secretory cargo together with early and late Golgi resident proteins tagged with three different fluorescence proteins. We have demonstrated that secretory cargo is indeed delivered within the Golgi by cisternal maturation. In addition, we have visualized details of secretory cargo trafficking in the Golgi, including formation of zones within a maturing cisterna, in which Golgi resident proteins are segregated, and movement of cargo between these zones. This protocol can be used for simultaneous three-color and four-dimensional observation of various phenomena in living cells, from yeast to higher plants and animals, at high spatiotemporal resolution.
0 Q&A 3757 Views Jun 20, 2020
Sarcomeres are extremely highly ordered macromolecular assemblies where proper structural organization is an absolute prerequisite to the functionality of these contractile units. Despite the wealth of information collected, the exact spatial arrangement of many of the H-zone and Z-disk proteins remained unknown. Recently, we developed a powerful nanoscopic approach to localize the sarcomeric protein components with a resolution well below the diffraction limit. The ease of sample preparation and the near crystalline structure of the Drosophila flight muscle sarcomeres make them ideally suitable for single molecule localization microscopy and structure averaging. Our approach allowed us to determine the position of dozens of H-zone and Z-disk proteins with a quasi-molecular, ~5-10 nm localization precision. The protocol described below provides an easy and reproducible method to prepare individual myofibrils for dSTORM imaging. In addition, it includes an in-depth description of a custom made and freely available software toolbox to process and quantitatively analyze the raw localization data.
0 Q&A 4916 Views Nov 20, 2019
Our mechanistic understanding of cell function depends on imaging biological processes in cells with molecular resolution. Super-resolution fluorescence microscopy plays a crucial role by reporting cellular ultrastructure with 20-30 nm resolution. However, this resolution is insufficient to image macro-molecular machinery at work. A path to improve resolution is to image under cryogenic conditions, which substantially increases the brightness of most fluorophores and preserves native ultrastructure much better than chemical fixatives. Cryogenic conditions are, however, underutilized because of the lack of compatible high numerical aperture (NA) objectives. Here we describe a protocol for the use of super-hemispherical solid immersion lenses (superSILs) to achieve super-resolution imaging at cryogenic temperatures with an effective NA of 2.17 and resolution of ~10 nm.
0 Q&A 5114 Views Dec 5, 2018
The Human Epidermal Growth Factor Receptor (HER) family of receptor tyrosine kinases consists of four, single pass, transmembrane receptor homologs (HER1-4) that act to regulate many critical processes in normal and tumor cells. HER2 is overexpressed in many tumors, and the deregulated proliferation of cancerous cells is driven by cooperation with its preferred receptor partner, HER3. The assessment of the in-situ organization of tagged HER2 and HER3 using super-resolution microscopy reveals quantitative Single Molecule Localization Microscopy (SMLM) as an ideal bioanalytical tool to characterize receptor clusters. Clustering of receptors is an important regulatory mechanism to prime cells to respond to stimuli so, to understand these processes, it is necessary to measure parameters such as numbers of clusters, cluster radii and the number of localizations per cluster for different perturbations. Previously, Fluorescence Localization Imaging with Photobleaching (FLImP), another nanoscale, single-molecule technique, characterized the oligomerization state of HER1 [or Epidermal Growth Factor Receptors (EGFR)] in cell membranes. To achieve an unprecedented resolution (< 5 nm) for inter-molecular separations in EGFR oligomers using FLImP, very few receptors are tagged, and so this method is unsuitable for measurements of whole receptor populations in cancer cells where receptors are frequently upregulated. Here, in order to detect all receptors involved in cluster formation, we saturate endogenous HER2 and HER3 membrane receptors with ligands at a 1:1 dye to protein ratio, in the presence or absence of therapeutic drugs (lapatinib or bosutinib). This is performed in the commonly used breast cancer cell line model SKBR3 cells, where there are ~1.6 million HER2 receptors/cell and 10,000-40,000 HER3 receptors/cell. The basal state of these receptors is studied using HER2- or HER3-specific Affibodies, and likewise, the active state is probed using the natural HER3 ligand, Neuregulin-beta1 (NRGβ1). Stochastic Optical Reconstruction Microscopy (STORM), one form of SMLM, was used here to image cells, which were chemically fixed to minimize image blurring and provide data (x and y coordinates and standard deviation of the measured localizations) for cluster analysis. Further analysis can also determine proportions of receptor colocalizations. Our findings show that lapatinib-bound HER2, complexed with HER3 via a non-canonical kinase dimer structure, induces higher order oligomers. We hypothesized that nucleation of receptors creates signaling platforms that explain the counterintuitive, increase in cell proliferation upon ligand binding, in the presence of the HER2-inhibitor lapatinib.



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