scholarly journals Automated highly multiplexed super-resolution imaging of protein nano-architecture in cells and tissues

2019 ◽  
Author(s):  
Maja Klevanski ◽  
Frank Herrmannsdoerfer ◽  
Varun Venkataramani ◽  
Steffen Sass ◽  
Mike Heilemann ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Protein Localization ◽  
Super Resolution ◽  
Protein Distribution ◽  
Tissue Samples ◽  
Target Proteins ◽  
Presynaptic Nerve Terminal ◽  
Solution Exchange ◽  
Staining Protocol ◽  
Cellular Compartments

ABSTRACTUnderstanding the nano-architecture of protein machines in diverse sub-cellular compartments remains a challenge despite rapid progress in super-resolution microscopy. While singlemolecule localization microscopy techniques allow the visualization and identification of cellular structures with near-molecular resolution, multiplex-labeling of tens of target proteins within the same sample has not yet been achieved routinely. However, single sample multiplexing is essential to detect patterns that threaten to get lost in multi-sample averaging. Here, we report maS3TORM (multiplexed automated serial staining stochastic optical reconstruction microscopy), a microscopy approach capable of fully automated 3D dSTORM imaging and solution exchange employing a re-staining protocol to achieve highly multiplexed protein localization within individual biological samples. We demonstrate 3D super-resolution images of 15 target proteins in single cultured cells and 16 targets in individual neuronal tissue samples with <10 nm localization precision. This allowed us to define novel nano-architectural features of protein distribution within the presynaptic nerve terminal.

scholarly journals Hypoxia elicits broad and systematic changes in protein subcellular localization

2011 ◽  
Vol 301 (4) ◽  
pp. C913-C928 ◽  
Author(s):  
Robert Michael Henke ◽  
Ranita Ghosh Dastidar ◽  
Ajit Shah ◽  
Daniela Cadinu ◽  
Xiao Yao ◽  
...  
Keyword(s):  
Protein Function ◽  
Protein Localization ◽  
Cell Periphery ◽  
Protein Distribution ◽  
Yeast Saccharomyces Cerevisiae ◽  
Transcriptional Regulatory ◽  
Chromatin Remodeling Complexes ◽  
Oxygen Signaling ◽  
Cellular Compartments ◽  
Control Protein

Oxygen provides a crucial energy source in eukaryotic cells. Hence, eukaryotes ranging from yeast to humans have developed sophisticated mechanisms to respond to changes in oxygen levels. Regulation of protein localization, like protein modifications, can be an effective mechanism to control protein function and activity. However, the contribution of protein localization in oxygen signaling has not been examined on a genomewide scale. Here, we examine how hypoxia affects protein distribution on a genomewide scale in the model eukaryote, the yeast Saccharomyces cerevisiae . We demonstrate, by live cell imaging, that hypoxia alters the cellular distribution of 203 proteins in yeast. These hypoxia-redistributed proteins include an array of proteins with important functions in various organelles. Many of them are nuclear and are components of key regulatory complexes, such as transcriptional regulatory and chromatin remodeling complexes. Under hypoxia, these proteins are synthesized and retained in the cytosol. Upon reoxygenation, they relocalize effectively to their normal cellular compartments, such as the nucleus, mitochondria, endoplasmic reticulum, and cell periphery. The resumption of the normal cellular locations of many proteins can occur even when protein synthesis is inhibited. Furthermore, we show that the changes in protein distribution induced by hypoxia follow a slower trajectory than those induced by reoxygenation. These results show that the regulation of protein localization is a common and potentially dominant mechanism underlying oxygen signaling and regulation. These results may have broad implications in understanding oxygen signaling and hypoxia responses in higher eukaryotes such as humans.

scholarly journals Novel approach for quantification of multiple immunofluorescent signals using histograms and 2D plot profiling of whole-section panoramic images

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Roko Duplancic ◽  
Darko Kero
Keyword(s):  
Histological Section ◽  
Cell Counting ◽  
Tissue Samples ◽  
Spatial Gradients ◽  
Multiple Markers ◽  
Novel Approach ◽  
Academic Settings ◽  
Panoramic Images ◽  
Multiple Regions ◽  
Staining Protocol

AbstractWe describe a novel approach for quantification and colocalization of immunofluorescence (IF) signals of multiple markers on high-resolution panoramic images of serial histological sections utilizing standard staining techniques and readily available software for image processing and analysis. Human gingiva samples stained with primary antibodies against the common leukocyte antigen CD45 and factors related to heparan sulfate glycosaminoglycans (HS GAG) were used. Expression domains and spatial gradients of IF signals were quantified by histograms and 2D plot profiles, respectively. The importance of histomorphometric profiling of tissue samples and IF signal thresholding is elaborated. This approach to quantification of IF staining utilizes pixel (px) counts and comparison of px grey value (GV) or luminance. No cell counting is applied either to determine the cellular content of a given histological section nor the number of cells positive to the primary antibody of interest. There is no selection of multiple Regions-Of-Interest (ROIs) since the entire histological section is quantified. Although the standard IF staining protocol is applied, the data output enables colocalization of multiple markers (up to 30) from a given histological sample. This can serve as an alternative for colocalization of IF staining of multiple primary antibodies based on repeating cycles of staining of the same histological section since those techniques require non standard staining protocols and sophisticated equipment that can be out of reach for small laboratories in academic settings. Combined with the data from ontological bases, this approach to quantification of IF enables creation of in silico virtual disease models.

Monitoring Drug Target Engagement in Cells and Tissues Using the Cellular Thermal Shift Assay

Science ◽  
2013 ◽  
Vol 341 (6141) ◽  
pp. 84-87 ◽  
Author(s):  
Daniel Martinez Molina ◽  
Rozbeh Jafari ◽  
Marina Ignatushchenko ◽  
Takahiro Seki ◽  
E. Andreas Larsson ◽  
...  
Keyword(s):  
Drug Target ◽  
Drug Transport ◽  
Drug Binding ◽  
Target Engagement ◽  
Tissue Samples ◽  
Target Proteins ◽  
Thermal Shift Assay ◽  
Cellular Thermal Shift Assay ◽  
Thermal Shift ◽  
In Cells

The efficacy of therapeutics is dependent on a drug binding to its cognate target. Optimization of target engagement by drugs in cells is often challenging, because drug binding cannot be monitored inside cells. We have developed a method for evaluating drug binding to target proteins in cells and tissue samples. This cellular thermal shift assay (CETSA) is based on the biophysical principle of ligand-induced thermal stabilization of target proteins. Using this assay, we validated drug binding for a set of important clinical targets and monitored processes of drug transport and activation, off-target effects and drug resistance in cancer cell lines, as well as drug distribution in tissues. CETSA is likely to become a valuable tool for the validation and optimization of drug target engagement.

scholarly journals Circumvention of common labeling artifacts using secondary nanobodies

2019 ◽  
Author(s):  
Shama Sograte-Idrissi ◽  
Thomas Schlichthaerle ◽  
Carlos J. Duque-Afonso ◽  
Mihai Alevra ◽  
Sebastian Strauss ◽  
...  
Keyword(s):  
Super Resolution ◽  
Light Sheet ◽  
Common Procedure ◽  
Tissue Samples ◽  
Localization Accuracy ◽  
Light Sheet Microscopy ◽  
Large Size ◽  
Resolution Imaging ◽  
Single Domain Antibodies ◽  
Secondary Antibodies

AbstractThe most common procedure to reveal the location of specific (sub)cellular elements in biological samples is via immunostaining followed by optical imaging. This is typically performed with target-specific primary antibodies (1.Abs), which are revealed by fluorophore-conjugated secondary antibodies (2.Abs). However, at high resolution this methodology can induce a series of artifacts due to the large size of antibodies, their bivalency, and their polyclonality. Here we use STED and DNA-PAINT super-resolution microscopy or light sheet microscopy on cleared tissue to show how monovalent secondary reagents based on camelid single-domain antibodies (nanobodies; 2.Nbs) attenuate these artifacts. We demonstrate that monovalent 2.Nbs have four additional advantages: 1) they increase localization accuracy with respect to 2.Abs; 2) they allow direct pre-mixing with 1.Abs before staining, reducing experimental time, and enabling the use of multiple 1.Abs from the same species; 3) they penetrate thick tissues efficiently; and 4) they avoid the artificial clustering seen with 2.Abs both in live and in poorly fixed samples. Altogether, this suggests that 2.Nbs are a valuable alternative to 2.Abs, especially when super-resolution imaging or staining of thick tissue samples are involved.

scholarly journals An efficient polymer cocktail-based transportation method for cartilage tissue, yielding chondrocytes with enhanced hyaline cartilage expression during in vitro culturing

2021 ◽  
Author(s):  
Shojiro Katoh ◽  
Hiroshi Yoshioka ◽  
Shoji Suzuki ◽  
Hiroyuki Nakajima ◽  
Masaru Iwasaki ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Hyaline Cartilage ◽  
Three Dimensional ◽  
Cartilage Tissue ◽  
Human Cartilage ◽  
Tissue Samples ◽  
Chondrocyte Implantation ◽  
Initial Processing ◽  
Regenerative Therapies

Chondrocytes are used in cell-based therapies such as autologous chondrocyte implantation (ACI) and matrix-associated cartilage implantation (MACI). To transport the cartilage tissue to the laboratory for in vitro culturing, phosphate-buffered saline (PBS), Euro-Collins solution (ECS) and Dulbecco Modified Eagle Medium (DMEM) are commonly employed at 4-8 deg C. In this study, eight samples of human cartilage biopsy tissues from elderly patients with severe osteoarthritis undergoing arthroscopy, which would otherwise have been discarded, were used. The cartilage tissue samples were compared to assess the cell yield between two transportation groups: i) a thermo-reversible gelation polymer (TGP) based method without cool preservation (~25 deg C) and ii) ECS transport at 4 deg C. These samples were subjected to in vitro culture in a two-dimensional (2D) monolayer for two weeks and subsequently in a three-dimensional (3D) TGP scaffold for six weeks. The cell count obtained from the tissues transported in TGP was higher (0.2 million cells) than those transported in ECS (0.08 million cells) both after initial processing and after in vitro culturing for 2 weeks in 2D (18 million cells compared with 10 million cells). In addition, mRNA quantification demonstrated significantly higher expression of Col2a1 and SOX-9 in 3D-TGP cultured cells and lower expression of COL1a1 in RT-PCR, characteristic of the hyaline cartilage phenotype, than in 2D culture. This study confirms that the TGP cocktail is suitable for both the transport of human cartilage tissue and for in vitro culturing to yield better-quality cells for use in regenerative therapies.

scholarly journals Microvillar cartography: a super-resolution single-molecule imaging method to map the positions of membrane proteins with respect to cellular surface topography

2020 ◽  
Author(s):  
Shirsendu Ghosh ◽  
Ronen Alon ◽  
Andres Alcover ◽  
Gilad Haran
Keyword(s):  
Cell Surface ◽  
Single Molecule ◽  
Cell Activation ◽  
Super Resolution ◽  
Cell Receptor ◽  
Specific Protein ◽  
Surface Proteins ◽  
Imaging Method ◽  
Protein Distribution ◽  
Membrane Topography

AbstractWe introduce Microvillar Cartography (MC), a method to map proteins on cellular surfaces with respect to the membrane topography. The surfaces of many cells are not smooth, but are rather covered with various protrusions such as microvilli. These protrusions may play key roles in multiple cellular functions, due to their ability to control the distribution of specific protein assemblies on the cell surface. Thus, for example, we have shown that the T-cell receptor and several of its proximal signaling proteins reside on microvilli, while others are excluded from these projections. These results have indicated that microvilli can function as key signaling hubs for the initiation of the immune response. MC has facilitated our observations of particular surface proteins and their specialized distribution on microvillar and non-microvillar compartments. MC combines membrane topography imaging, using variable-angle total internal microscopy, with stochastic localization nanoscopy, which generates deep sub-diffraction maps of protein distribution. Since the method is based on light microscopy, it avoids some of the pitfalls inherent to electron-microscopy-based techniques, such as dehydration, carbon coating and immunogold clustering, and is amenable to future developments involving e.g. live-cell imaging. This Protocol details the procedures we developed for MC, which can be readily adopted to study a broad range of cell surface molecules and dissect their distribution within distinct surface assemblies under multiple cell activation states.

scholarly journals Enhanced amyloid-β generation by γ-secretase complex in DRM microdomains with reduced cholesterol levels

2019 ◽  
Vol 29 (3) ◽  
pp. 382-393 ◽  
Author(s):  
Saori Hata ◽  
Anqi Hu ◽  
Yi Piao ◽  
Tadashi Nakaya ◽  
Hidenori Taru ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Late Onset ◽  
Senile Plaques ◽  
Amyloid Β ◽  
Cholesterol Content ◽  
Amyloid Β Protein ◽  
Tissue Samples ◽  
Β Protein ◽  
Aβ Generation ◽  
Carboxy Terminal

Abstract A neuropathologic hallmark of Alzheimer’s disease (AD) is the presence of senile plaques that contain neurotoxic amyloid-β protein (Aβ) species, which are generated by the cleavage of amyloid β-protein precursor by secretases such as the γ-secretase complex, preferentially located in detergent-resistant membrane (DRM) regions and comprising endoproteolysed amino- and carboxy-terminal fragments of presenilin, nicastrin, anterior pharynx defective 1 and presenilin enhancer 2. Whereas some of familial AD patients harbor causative PSEN mutations that lead to more generation of neurotoxic Aβ42, the contribution of Aβ generation to sporadic/late-onset AD remains unclear. We found that the carboxy-terminal fragment of presenilin 1 was redistributed from DRM regions to detergent-soluble membrane (non-DRM) regions in brain tissue samples from individuals with sporadic AD. DRM fractions from AD brain sample had the ability to generate significantly more Aβ and had a lower cholesterol content than DRM fractions from non-demented control subjects. We further demonstrated that lowering the cholesterol content of DRM regions from cultured cells contributed to the redistribution of γ-secretase components and Aβ production. Taken together, the present analyses suggest that the lowered cholesterol content in DRM regions may be a cause of sporadic/late-onset AD by enhancing overall Aβ generation.

scholarly journals Super-resolution microscopy can identify specific protein distribution patterns in platelets incubated with cancer cells

Nanoscale ◽  
2019 ◽  
Vol 11 (20) ◽  
pp. 10023-10033 ◽  
Author(s):  
Jan Bergstrand ◽  
Lei Xu ◽  
Xinyan Miao ◽  
Nailin Li ◽  
Ozan Öktem ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Dictionary Learning ◽  
Super Resolution ◽  
Distribution Patterns ◽  
Specific Protein ◽  
Protein Distribution ◽  
Activated Platelets ◽  
Resolution Imaging ◽  
Super Resolution Microscopy

Super-resolution imaging of P-selectin in platelets together with dictionary learning allow specifically activated platelets to be identified in an automatic objective manner.

scholarly journals Parallel Acquisition of Plasma Membrane Ultrastructure and Cytosolic Protein Localisation in Cultured Cells via Correlated Immunogold SEM

Cells ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 1329
Author(s):  
Isabell Begemann ◽  
Ulrike Keller ◽  
Harald Nüsse ◽  
Jürgen Klingauf ◽  
Milos Galic
Keyword(s):  
Plasma Membrane ◽  
Cultured Cells ◽  
Intact Cell ◽  
Protein Localization ◽  
Integrated Approach ◽  
Detection Methods ◽  
Parallel Acquisition ◽  
Cytosolic Protein ◽  
Correlated Information ◽  
Membrane Ultrastructure

Scanning electron microscopy (SEM) takes advantage of distinct detectors to visualise secondary and back-scattering electrons. Here, we report an integrated approach that relies on these two detection methods to simultaneously acquire correlated information on plasma membrane topography and curvature-sensitive cytosolic protein localization in intact cell samples. We further provide detailed preparation and staining protocols, as well as a thorough example-based discussion for imaging optimisation. Collectively, the presented method enables rapid and precise analysis of cytosolic proteins adjacent to cellular membranes with a resolution of ~100 nm, without time-consuming preparations or errors induced by sequential visualisation present in fluorescence-based correlative approaches.

scholarly journals Integrating Protein Localization with Automated Signaling Pathway Reconstruction

2019 ◽  
Author(s):  
Ibrahim Youssef ◽  
Jeffrey Law ◽  
Anna Ritz
Keyword(s):  
Signal Transduction ◽  
Signaling Pathways ◽  
Protein Interactions ◽  
Cellular Localization ◽  
Protein Localization ◽  
Dynamic Program ◽  
Reconstruction Algorithms ◽  
Ppi Networks ◽  
Cellular Compartments ◽  
Localization Information

AbstractUnderstanding cellular responses via signal transduction is a core focus in systems biology. Tools to automatically reconstruct signaling pathways from protein-protein interactions (PPIs) can help biologists generate testable hypotheses about signaling. However, automatic reconstruction of signaling pathways suffers from many interactions with the same confidence score leading to many equally good candidates. Further, some reconstructions are biologically misleading due to ignoring protein localization information. We proposeLocPL, a method to improve the automatic reconstruction of signaling pathways from PPIs by incorporating information about protein localization in the reconstructions. The method relies on a dynamic program to ensure that the proteins in a reconstruction are localized in cellular compartments that are consistent with signal transduction from the membrane to the nucleus.LocPLand existing reconstruction algorithms are applied to two PPI networks and assessed using both global and local definitions of accuracy.LocPLproduces more accurate and biologically meaningful reconstructions on a versatile set of signaling pathways.LocPLis a powerful tool to automatically reconstruct signaling pathways from PPIs that leverages cellular localization information about proteins. The underlying dynamic program and signaling model are flexible enough to study cellular signaling under different settings of signaling flow across the cellular compartments.

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