methods-for-visualizing-intracellular-organelles

Methods For Visualizing Intracellular Organelles

ARTICLES DISCUSSED:
Aishwarya, R. et al. Visualizing subcellular localization of a protein in the heart using quantum dots-mediated immuno-labeling ...

antibody-uptake-assay-for-tracking-notchdelta-endocytosis-during

Antibody Uptake Assay for Tracking Notch/Delta Endocytosis During the Asymmetric Division of Zebrafish Radial Glia Progenitors

Asymmetric cell division (ACD), which produces two daughter cells of different fates, is fundamental for generating cellular diversity. In the developing ...

Chan Zuckerberg Biohub

University of California, San Francisco

University of California San Francisco

Foreign antigens are presented by antigen-presenting cells in the presence of abundant endogenous peptides that are nonstimulatory to the T cell. In mouse T cells, endogenous, nonstimulatory peptides have been shown to enhance responses to specific peptide antigens, a phenomenon termed coagonism. However, whether coagonism also occurs in human T cells is unclear, and the molecular mechanism of coagonism is still under debate since CD4 and CD8 coagonism requires different interactions. Here we show that the nonstimulatory, HIV-derived peptide GAG enhances a specific human cytotoxic T lymphocyte response to HBV-derived epitopes presented by HLA-A*02:01. Coagonism in human T cells requires the CD8 coreceptor, but not T-cell receptor (TCR) binding to the nonstimulatory peptide-MHC. Coagonists enhance the phosphorylation and recruitment of several molecules involved in the TCR-proximal signaling pathway, suggesting that coagonists promote T-cell responses to antigenic pMHC by amplifying TCR-proximal signaling.

Nonstimulatory peptide-MHC enhances human T-cell antigen-specific responses by amplifying proximal TCR signaling.

In the developing embryos, the cortical polarity regulator Par-3 is critical for establishing Notch signaling asymmetry between daughter cells during asymmetric cell division (ACD). How cortically localized Par-3 establishes asymmetric Notch activity in the nucleus is not understood. Here, using in vivo time-lapse imaging of mitotic radial glia progenitors in the developing zebrafish forebrain, we uncover that during horizontal ACD along the anteroposterior embryonic axis, endosomes containing the Notch ligand DeltaD (Dld) move toward the cleavage plane and preferentially segregate into the posterior (subsequently basal) Notch daughter. This asymmetric segregation requires the activity of Par-3 and dynein motor complex. Using label retention expansion microscopy, we further detect Par-3 in the cytosol colocalizing the dynein light intermediate chain 1 (Dlic1) onto Dld endosomes. Par-3, Dlic1, and Dld are associated in protein complexes in vivo. Our data reveal an unanticipated mechanism by which cytoplasmic Par-3 directly polarizes Notch signaling components during ACD.

Polarized endosome dynamics engage cytoplasmic Par-3 that recruits dynein during asymmetric cell division.

Parkinson's disease (PD) is a common neurodegenerative disorder without effective disease-modifying therapeutics. Here, we establish a chemogenetic dopamine (DA) neuron ablation model in larval zebrafish with mitochondrial dysfunction and robustness suitable for high-content screening. We use this system to conduct an in vivo DA neuron imaging-based chemical screen and identify the Renin-Angiotensin-Aldosterone System (RAAS) inhibitors as significantly neuroprotective. Knockdown of the angiotensin receptor 1 () in DA neurons reveals a cell-autonomous mechanism of neuroprotection. DA neuron-specific RNA-seq identifies mitochondrial pathway gene expression that is significantly restored by RAAS inhibitor treatment. The neuroprotective effect of RAAS inhibitors is further observed in a zebrafish Gaucher disease model and -deficient PD model. Finally, examination of clinical data reveals a significant effect of RAAS inhibitors in delaying PD progression. Our findings reveal the therapeutic potential and mechanisms of targeting the RAAS pathway for neuroprotection and demonstrate a salient approach that bridges basic science to translational medicine.

A zebrafish screen reveals Renin-angiotensin system inhibitors as neuroprotective via mitochondrial restoration in dopamine neurons.

The promise of single-objective light-sheet microscopy is to combine the convenience of standard single-objective microscopes with the speed, coverage, resolution and gentleness of light-sheet microscopes. We present DaXi, a single-objective light-sheet microscope design based on oblique plane illumination that achieves: (1) a wider field of view and high-resolution imaging via a custom remote focusing objective; (2) fast volumetric imaging over larger volumes without compromising image quality or necessitating tiled acquisition; (3) fuller image coverage for large samples via multi-view imaging and (4) higher throughput multi-well imaging via remote coverslip placement. Our instrument achieves a resolution of 450 nm laterally and 2 μm axially over an imaging volume of 3,000 × 800 × 300 μm. We demonstrate the speed, field of view, resolution and versatility of our instrument by imaging various systems, including Drosophila egg chamber development, zebrafish whole-brain activity and zebrafish embryonic development - up to nine embryos at a time.

DaXi-high-resolution, large imaging volume and multi-view single-objective light-sheet microscopy.

Cytokines are powerful immune modulators that initiate signaling through receptor dimerization, but natural cytokines have structural limitations as therapeutics. We present a strategy to discover cytokine surrogate agonists by using modular ligands that exploit induced proximity and receptor dimer geometry as pharmacological metrics amenable to high-throughput screening. Using VHH and scFv to human interleukin-2/15, type-I interferon, and interleukin-10 receptors, we generated combinatorial matrices of single-chain bispecific ligands that exhibited diverse spectrums of functional activities, including potent inhibition of SARS-CoV-2 by surrogate interferons. Crystal structures of IL-2R:VHH complexes revealed that variation in receptor dimer geometries resulted in functionally diverse signaling outputs. This modular platform enabled engineering of surrogate ligands that compelled assembly of an IL-2R/IL-10R heterodimer, which does not naturally exist, that signaled through pSTAT5 on T and natural killer (NK) cells. This "cytokine med-chem" approach, rooted in principles of induced proximity, is generalizable for discovery of diversified agonists for many ligand-receptor systems.

Facile discovery of surrogate cytokine agonists.

Adoptive cell therapy using engineered T cell receptors (TCRs) is a promising approach for targeting cancer antigens, but tumor-reactive TCRs are often weakly responsive to their target ligands, peptide-major histocompatibility complexes (pMHCs). Affinity-matured TCRs can enhance the efficacy of TCR-T cell therapy but can also cross-react with off-target antigens, resulting in organ immunopathology. We developed an alternative strategy to isolate TCR mutants that exhibited high activation signals coupled with low-affinity pMHC binding through the acquisition of catch bonds. Engineered analogs of a tumor antigen MAGE-A3-specific TCR maintained physiological affinities while exhibiting enhanced target killing potency and undetectable cross-reactivity, compared with a high-affinity clinically tested TCR that exhibited lethal cross-reactivity with a cardiac antigen. Catch bond engineering is a biophysically based strategy to tune high-sensitivity TCRs for T cell therapy with reduced potential for adverse cross-reactivity.

Tuning T cell receptor sensitivity through catch bond engineering.

Asymmetric cell division (ACD) is fundamental for balancing cell proliferation and differentiation in metazoans. During active neurogenesis in the developing zebrafish forebrain, radial glia progenitors (RGPs) mainly undergo ACD to produce one daughter with high activity of Delta/Notch signaling (proliferative cell fate) and another daughter with low Delta/Notch signaling (differentiative cell fate). The cell polarity protein partitioning-defective 3 (Par-3) is critical for regulating this process. To understand how polarized Par-3 on the cell cortex can lead to differential Notch activity in the nuclei of daughter cells, we combined an anti-Delta D (Dld) -atto 647N antibody uptake assay with label retention expansion microscopy (LR-ExM), to obtain high resolution immunofluorescent images of Par-3, dynein light intermediate chain 1 (Dlic1), and Dld endosomes in mitotic RGPs. We then developed a protocol for analyzing the colocalization of Par-3, Dlic1, and endosomal DeltaD, using JACoP (Just Another Co-localization Plugin) in ImageJ software (Bolte and Cordelières, 2006). Through such analyses, we have shown that cytosolic Par-3 is associated with Dlic1 on Dld endosomes. Our work demonstrates a direct involvement of Par-3 in dynein-mediated polarized transport of Notch signaling endosomes. This bio-protocol may be generalizable for analysis of protein co-localization in any cryosectioned and immunostained tissue samples.

Colocalization Analysis for Cryosectioned and Immunostained Tissue Samples with or without Label Retention Expansion Microscopy (LR-ExM) by JACoP.

Adaptive immunity relies on T lymphocytes that use αβ T cell receptors (TCRs) to discriminate among peptides presented by major histocompatibility complex molecules (pMHCs). Identifying pMHCs capable of inducing robust T cell responses will not only enable a deeper understanding of the mechanisms governing immune responses but could also have broad applications in diagnosis and treatment. T cell recognition of sparse antigenic pMHCs in vivo relies on biomechanical forces. However, in vitro screening methods test potential pMHCs without force and often at high (nonphysiological) pMHC densities and thus fail to predict potent agonists in vivo. Here, we present a technology termed BATTLES (biomechanically assisted T cell triggering for large-scale exogenous-pMHC screening) that uses biomechanical force to initiate T cell triggering for peptides and cells in parallel. BATTLES displays candidate pMHCs on spectrally encoded beads composed of a thermo-responsive polymer capable of applying shear loads to T cells, facilitating exploration of the force- and sequence-dependent landscape of T cell responses. BATTLES can be used to explore basic T cell mechanobiology and T cell-based immunotherapies.

ArticleTotal : 2	Year
Publication title Cited by 4	2023
Publication title Cited by 5	2023

Article	Year
Nonstimulatory peptide-MHC enhances human T-cell antigen-specific responses by amplifying proximal TCR signaling. Nature communications\| PubMed ID: 30006605	2018
Polarized endosome dynamics engage cytoplasmic Par-3 that recruits dynein during asymmetric cell division. Science advances\| PubMed ID: 34117063	2021
A zebrafish screen reveals Renin-angiotensin system inhibitors as neuroprotective via mitochondrial restoration in dopamine neurons. eLife\| PubMed ID: 34550070	2021
DaXi-high-resolution, large imaging volume and multi-view single-objective light-sheet microscopy. Nature methods\| PubMed ID: 35314838	2022
Facile discovery of surrogate cytokine agonists. Cell\| PubMed ID: 35325595	2022
Tuning T cell receptor sensitivity through catch bond engineering. Science (New York, N.Y.)\| PubMed ID: 35389803	2022
Colocalization Analysis for Cryosectioned and Immunostained Tissue Samples with or without Label Retention Expansion Microscopy (LR-ExM) by JACoP. Bio-protocol\| PubMed ID: 35592606	2022
A bead-based method for high-throughput mapping of the sequence- and force-dependence of T cell activation. Nature methods\| PubMed ID: 36064771	2022

Xiang Zhao

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