scholarly journals RIM-Binding Protein 2 organizes Ca2+ channel topography and regulates release probability and vesicle replenishment at a fast central synapse

2021 ◽  
Author(s):  
Tanvi Butola ◽  
Theocharis Alvanos ◽  
Anika Hintze ◽  
Peter Koppensteiner ◽  
David Kleindienst ◽  
...  
Keyword(s):  
Binding Protein ◽  
Light And Electron Microscopy ◽  
Super Resolution ◽  
Auditory Pathway ◽  
Nerve Fibers ◽  
Release Probability ◽  
High Release ◽  
Train Stimulation ◽  
Bushy Cells

RIM-Binding Protein 2 (RIM-BP2) is a multi-domain protein of the presynaptic active zone (AZ). By binding to Rab-interacting protein (RIM), bassoon and voltage-gated Ca2+ channels (CaV), it is considered to be a central organizer of the topography of CaV and release sites of synaptic vesicles (SVs) at the AZ. Here, we investigated the role of RIM-BP2 at the endbulb of Held synapse of auditory nerve fibers with bushy cells of the cochlear nucleus, a fast relay of the auditory pathway with high release probability. Disruption of RIM-BP2 lowered release probability altering short-term plasticity and reduced evoked excitatory postsynaptic currents (EPSCs). Analysis of SV pool dynamics during high frequency train stimulation indicated a reduction of SVs with high release probability but an overall normal size of the readily releasable SV pool (RRP). The Ca2+-dependent fast component of SV replenishment after RRP depletion was slowed. Augmenting Ca2+ influx by adding extracellular Ca2+ restored release probability but not EPSC amplitude, and uncovered an impairment of SV replenishment during train stimulation. Ultrastructural analysis by super-resolution light and electron microscopy revealed an impaired topography of presynaptic CaV and a reduction of docked and membrane-proximal SVs at the AZ. We conclude that RIM-BP2 organizes the topography of CaV, and promotes SV tethering and docking. This way RIM-BP2 is critical for establishing a high initial release probability as required to reliably signal sound onset information that we found to be degraded in bushy cells of RIM-BP2-deficient mice in vivo.

scholarly journals Application of Fluorescent Proteins for Functional Dissection of the Drosophila Visual System

2021 ◽  
Vol 22 (16) ◽  
pp. 8930
Author(s):  
Thomas K. Smylla ◽  
Krystina Wagner ◽  
Armin Huber
Keyword(s):  
Protein Transport ◽  
Fluorescent Proteins ◽  
Protein Localization ◽  
Expression Patterns ◽  
Light And Electron Microscopy ◽  
Super Resolution ◽  
Visual Signal ◽  
Genetic Screens ◽  
Drosophila Eye

The Drosophila eye has been used extensively to study numerous aspects of biological systems, for example, spatio-temporal regulation of differentiation, visual signal transduction, protein trafficking and neurodegeneration. Right from the advent of fluorescent proteins (FPs) near the end of the millennium, heterologously expressed fusion proteins comprising FPs have been applied in Drosophila vision research not only for subcellular localization of proteins but also for genetic screens and analysis of photoreceptor function. Here, we summarize applications for FPs used in the Drosophila eye as part of genetic screens, to study rhodopsin expression patterns, subcellular protein localization, membrane protein transport or as genetically encoded biosensors for Ca2+ and phospholipids in vivo. We also discuss recently developed FPs that are suitable for super-resolution or correlative light and electron microscopy (CLEM) approaches. Illustrating the possibilities provided by using FPs in Drosophila photoreceptors may aid research in other sensory or neuronal systems that have not yet been studied as well as the Drosophila eye.

scholarly journals Platelet Imaging

Platelets ◽  
2020 ◽  
Author(s):  
Zachary A. Matthay ◽  
Lucy Zumwinkle Kornblith
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Electron Tomography ◽  
Thrombus Formation ◽  
Light And Electron Microscopy ◽  
Super Resolution ◽  
Experimental Systems ◽  
Health And Disease ◽  
Fluorescence Light

The knowledge gained through imaging platelets has formed the backbone of our understanding of their biology in health and disease. Early investigators relied on conventional light microscopy with limited resolution and were primarily able to identify the presence and basic morphology of platelets. The advent of high resolution technologies, in particular, electron microscopy, accelerated our understanding of the dynamics of platelet ultrastructure dramatically. Further refinements and improvements in our ability to localize and reliably identify platelet structures have included the use of immune-labeling techniques, correlative-fluorescence light and electron microscopy, and super-resolution microscopies. More recently, the expanded development and application of intravital microscopy in animal models has enhanced our knowledge of platelet functions and thrombus formation in vivo, as these experimental systems most closely replicate native biological environments. Emerging improvements in our ability to characterize platelets at the ultrastructural and organelle levels include the use of platelet cryogenic electron tomography with quantitative, unbiased imaging analysis, and the ability to genetically label platelet features with electron dense markers for analysis by electron microscopy.

Solving the mechanisms responsible for organelle behavior in vivo by same-cell correlative light and electron microscopy

1992 ◽  
Vol 50 (1) ◽  
pp. 14-15
Author(s):  
Conly L. Rieder
Keyword(s):  
Electron Microscopy ◽  
Quantitative Analysis ◽  
Light Microscopy ◽  
Living Cell ◽  
Light And Electron Microscopy ◽  
Time Behavior ◽  
Dynamic Interactions ◽  
Positive Identification ◽  
Cellular Components

The behavior of many cellular components, and their dynamic interactions, can be characterized in the living cell with considerable spatial and temporal resolution by video-enhanced light microscopy (video-LM). Indeed, under the appropriate conditions video-LM can be used to determine the real-time behavior of organelles ≤ 25-nm in diameter (e.g., individual microtubules—see). However, when pushed to its limit the structures and components observed within the cell by video-LM cannot be resolved nor necessarily even identified, only detected. Positive identification and a quantitative analysis often requires the corresponding electron microcopy (EM).

Programmable in vitro Co-Encapsidation of Guest Proteins Inside Protein Nanocages

2018 ◽  
Author(s):  
Noor H. Dashti ◽  
Rufika S. Abidin ◽  
Frank Sainsbury
Keyword(s):  
Self Assembly ◽  
Mammalian Cells ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Super Resolution ◽  
Cell Engineering ◽  
Particle Analysis ◽  
Protein Cages

Bioinspired self-sorting and self-assembling systems using engineered versions of natural protein cages have been developed for biocatalysis and therapeutic delivery. The packaging and intracellular delivery of guest proteins is of particular interest for both <i>in vitro</i> and <i>in vivo</i> cell engineering. However, there is a lack of platforms in bionanotechnology that combine programmable guest protein encapsidation with efficient intracellular uptake. We report a minimal peptide anchor for <i>in vivo</i> self-sorting of cargo-linked capsomeres of the Murine polyomavirus (MPyV) major coat protein that enables controlled encapsidation of guest proteins by <i>in vitro</i> self-assembly. Using Förster resonance energy transfer (FRET) we demonstrate the flexibility in this system to support co-encapsidation of multiple proteins. Complementing these ensemble measurements with single particle analysis by super-resolution microscopy shows that the stochastic nature of co-encapsidation is an overriding principle. This has implications for the design and deployment of both native and engineered self-sorting encapsulation systems and for the assembly of infectious virions. Taking advantage of the encoded affinity for sialic acids ubiquitously displayed on the surface of mammalian cells, we demonstrate the ability of self-assembled MPyV virus-like particles to mediate efficient delivery of guest proteins to the cytosol of primary human cells. This platform for programmable co-encapsidation and efficient cytosolic delivery of complementary biomolecules therefore has enormous potential in cell engineering.

scholarly journals C4b-binding protein α-chain enhances antitumor immunity by facilitating the accumulation of tumor-infiltrating lymphocytes in the tumor microenvironment in pancreatic cancer

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Kosuke Sasaki ◽  
Shigetsugu Takano ◽  
Satoshi Tomizawa ◽  
Yoji Miyahara ◽  
Katsunori Furukawa ◽  
...  
Keyword(s):  
T Cell ◽  
Tumor Microenvironment ◽  
Binding Protein ◽  
Combined Treatment ◽  
Tumor Infiltrating Lymphocytes ◽  
Pdac Cell ◽  
Α Chain ◽  
Infiltrating Lymphocytes

Abstract Background Recent studies indicate that complement plays pivotal roles in promoting or suppressing cancer progression. We have previously identified C4b-binding protein α-chain (C4BPA) as a serum biomarker for the early detection of pancreatic ductal adenocarcinoma (PDAC). However, its mechanism of action remains unclear. Here, we elucidated the functional roles of C4BPA in PDAC cells and the tumor microenvironment. Methods We assessed stromal C4BPA, the C4BPA binding partner CD40, and the number of CD8+ tumor-infiltrating lymphocytes in resected human PDAC tissues via immunohistochemical staining. The biological functions of C4BPA were investigated in peripheral blood mononuclear cells (PBMCs) and human PDAC cell lines. Mouse C4BPA (mC4BPA) peptide, which is composed of 30 amino acids from the C-terminus and binds to CD40, was designed for further in vitro and in vivo experiments. In a preclinical experiment, we assessed the efficacy of gemcitabine plus nab-paclitaxel (GnP), dual immune checkpoint blockades (ICBs), and mC4BPA peptide in a mouse orthotopic transplantation model. Results Immunohistochemical analysis revealed that high stromal C4BPA and CD40 was associated with favorable PDAC prognosis (P=0.0005). Stromal C4BPA strongly correlated with the number of CD8+ tumor-infiltrating lymphocytes (P=0.001). In in vitro experiments, flow cytometry revealed that recombinant human C4BPA (rhC4BPA) stimulation increased CD4+ and CD8+ T cell numbers in PBMCs. rhC4BPA also promoted the proliferation of CD40-expressing PDAC cells. By contrast, combined treatment with gemcitabine and rhC4BPA increased PDAC cell apoptosis rate. mC4BPA peptide increased the number of murine T lymphocytes in vitro and the number of CD8+ tumor-infiltrating lymphocytes surrounding PDAC tumors in vivo. In a preclinical study, GnP/ICBs/mC4BPA peptide treatment, but not GnP treatment, led to the accumulation of a greater number of CD8+ T cells in the periphery of PDAC tumors and to greater tumor regression than did control treatment. Conclusions These findings demonstrate that the combination of GnP therapy with C4BPA inhibits PDAC progression by promoting antitumor T cell accumulation in the tumor microenvironment.

scholarly journals Gran1: A Granulysin-Derived Peptide with Potent Activity against Intracellular Mycobacterium tuberculosis

2021 ◽  
Vol 22 (16) ◽  
pp. 8392
Author(s):  
Reiner Noschka ◽  
Fanny Wondany ◽  
Gönül Kizilsavas ◽  
Tanja Weil ◽  
Gilbert Weidinger ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Mycobacterium Tuberculosis ◽  
Developmental Toxicity ◽  
Super Resolution ◽  
In Vivo Models ◽  
Large Size ◽  
Acid Fragment ◽  
Powerful Approach ◽  
Target Effects

Granulysin is an antimicrobial peptide (AMP) expressed by human T-lymphocytes and natural killer cells. Despite a remarkably broad antimicrobial spectrum, its implementation into clinical practice has been hampered by its large size and off-target effects. To circumvent these limitations, we synthesized a 29 amino acid fragment within the putative cytolytic site of Granulysin (termed “Gran1”). We evaluated the antimicrobial activity of Gran1 against the major human pathogen Mycobacterium tuberculosis (Mtb) and a panel of clinically relevant non-tuberculous mycobacteria which are notoriously difficult to treat. Gran1 efficiently inhibited the mycobacterial proliferation in the low micro molar range. Super-resolution fluorescence microscopy and scanning electron microscopy indicated that Gran1 interacts with the surface of Mtb, causing lethal distortions of the cell wall. Importantly, Gran1 showed no off-target effects (cytokine release, chemotaxis, cell death) in primary human cells or zebrafish embryos (cytotoxicity, developmental toxicity, neurotoxicity, cardiotoxicity). Gran1 was selectively internalized by macrophages, the major host cell of Mtb, and restricted the proliferation of the pathogen. Our results demonstrate that the hypothesis-driven design of AMPs is a powerful approach for the identification of small bioactive compounds with specific antimicrobial activity. Gran1 is a promising component for the design of AMP-containing nanoparticles with selective activity and favorable pharmacokinetics to be pushed forward into experimental in vivo models of infectious diseases, most notably tuberculosis.

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