Endogenous tagging of Munc13-1 with a SNAP tag as a tool for monitoring synapse nanoarchitecture DOI Creative Commons

Maria Kowald,

Sylvestre P. J. T. Bachollet, Fritz Benseler

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Oct. 10, 2024

ABSTRACT Synaptic function is governed by highly regulated protein machineries, whose abundance and spatial localization change continually. Studies to determine the dynamic changes in synaptic proteins nanoarchitecture typically rely on immunolabeling, or expression of fluorescent proteins. The former employs chemical fluorophores signal amplification, but requires fixation. latter enables monitoring live microscopy, uses suboptimal fluorophores. Self-labeling tags have been introduced combine advantages these two approaches, here we introduce a knock-in mouse line where essential presynaptic Munc13-1 endogenously fused self-labeling SNAP tag. We demonstrate efficient Munc13-1-SNAP labeling fixed neurons brain sections various dyes, as well novel far-red cell impermeable compound, SBG-SiR-d12. characterize SBG-SiR-d12 highly-efficient dye for SNAP-tag extracellular epitopes, intracellular permeabilized tissue. Finally, show that can be labeled living monitored through live-cell imaging using confocal- super resolution microscopy. conclude Unc13a useful tool analysis nanoarchitectural dynamics, with potential wide adoption.

Language: Английский

GLP1R Attenuates Sympathetic Response to High Glucose via Carotid Body Inhibition DOI Creative Commons
Audrys G. Pauža, Pratik Thakkar, Tatjana Tasić

et al.

Circulation Research, Journal Year: 2022, Volume and Issue: 130(5), P. 694 - 707

Published: Feb. 1, 2022

Background: Aberrant sympathetic nerve activity exacerbates cardiovascular risk in hypertension and diabetes, which are common comorbidities, yet clinically remains poorly controlled. The hypertensive diabetic state is associated with increased reflex sensitivity tonic drive from the peripheral chemoreceptors, cause of unknown. We have previously shown to be critically dependent on carotid body (CB) input spontaneously rat, a model that also exhibits number traits. CB overstimulation by insulin leptin has been similarly implicated development metabolic syndrome obesity. Thus, we hypothesized (spontaneously rat), sensitized altered signaling causing excessive levels dysfunctional regulation. Methods: Using hypothesis-free RNA-seq approach, investigated potential molecular targets energy metabolism mediating sensitization its regulation outflow experimental hypertension. Identified were characterized using functional techniques assessing chemoreflex situ vivo. Results: discovered GLP1R (glucagon-like peptide-1 receptor) expression CBs rat human showed decreased linked hyperactivity rats cardiometabolic disease. demonstrate localized chemosensory cells, while targeted administration agonist lowered basal discharge attenuated chemoreflex-evoked blood pressure responses. Importantly, hyperglycemia-induced overactivity abolished activation suggesting role homeostatic response high glucose. Conclusions: show GLP1 peptide-1) modulates acting CB, supporting this organ as multimodal receptor. Our findings pinpoint for ameliorating agonists hypertensive-diabetic condition.

Language: Английский

Citations

61
N-Methyl deuterated rhodamines for protein labelling in sensitive fluorescence microscopy DOI Creative Commons
Kilian Roßmann, Kerem Can Akkaya, Pascal Poc

et al.

Chemical Science, Journal Year: 2022, Volume and Issue: 13(29), P. 8605 - 8617

Published: Jan. 1, 2022

Rhodamine fluorophores are setting benchmarks in fluorescence microscopy. Herein, we report the deuterium (d12) congeners of tetramethyl(silicon)rhodamine, obtained by isotopic labelling four methyl groups, show improved photophysical parameters (

Language: Английский

Citations

29
Revealing the tissue-level complexity of endogenous glucagon-like peptide-1 receptor expression and signaling DOI Creative Commons
Julia Ast, Daniela Nasteska, Nicholas H. F. Fine

et al.

Nature Communications, Journal Year: 2023, Volume and Issue: 14(1)

Published: Jan. 18, 2023

Abstract The glucagon-like peptide-1 receptor (GLP1R) is a class B G protein-coupled (GPCR) involved in glucose homeostasis and food intake. GLP1R agonists (GLP1RA) are widely used the treatment of diabetes obesity, yet visualizing endogenous localization, organization dynamics GPCR has so far remained out reach. In present study, we generate mice harboring an enzyme self-label genome-edited into Glp1r locus. We also rationally design test various fluorescent dyes, spanning cyan to far-red wavelengths, for labeling performance tissue. By combining these technologies, show that can be specifically sensitively detected primary tissue using multiple colors. Longitudinal analysis reveals heterogeneous recruitment neighboring cell subpopulations signaling trafficking, with differences observed between GLP1RA classes dual agonists. At nanoscopic level, GLP1Rs found possess higher organization, undergoing GLP1RA-dependent membrane diffusion. Together, results utility self-labels visualization interrogation proteins, provide insight biology cells

Language: Английский

Citations

18
Fluorogenic Chemical Probes for Wash-free Imaging of Cell Membrane Damage in Ferroptosis, Necrosis, and Axon Injury DOI

Philipp Mauker,

Daniela Beckmann,

Annabel Kitowski

et al.

Journal of the American Chemical Society, Journal Year: 2024, Volume and Issue: unknown

Published: April 9, 2024

Selectively labeling cells with damaged membranes is needed not only for identifying dead in culture, but also imaging membrane barrier dysfunction pathologies vivo. Most permeability stains are permanently colored or fluorescent dyes that need washing to remove their non-uptaken extracellular background and reach good image contrast. Others DNA-binding environment-dependent fluorophores, which lack design modularity, have potential toxicity, can detect permeabilization of cell volumes containing a nucleus (i.e., cannot delineate vivo nor non-nucleated types compartments). Here, we develop modular fluorogenic probes reveal the whole cytosolic volume cells, near-zero fluorescence so no needed. We identify specific disulfonated probe type enters membranes, then enzymatically activated marks them. The esterase MDG1 reliable tool live been permeabilized by biological, biochemical, physical damage, it be used multicolor microscopy. confirm modularity this approach adapting improved hydrolytic stability, as redox MDG2. conclude showing unique performance MDG revealing axonal damage (which DNA fluorogens achieve) discriminating on cell-by-cell basis embryos thus provides powerful tools wash-free indicates how designs may adapted selective delivery drug cargoes these cells: offering an outlook from diagnosis toward therapy membrane-compromised disease.

Language: Английский

Citations

8
Emerging modes of regulation of neuromodulatory G protein-coupled receptors DOI
Alberto J. Gonzalez-Hernandez, Hermany Munguba, Joshua Levitz

et al.

Trends in Neurosciences, Journal Year: 2024, Volume and Issue: 47(8), P. 635 - 650

Published: June 12, 2024

Language: Английский

Citations

6
Ligand-Specific Factors Influencing GLP-1 Receptor Post-Endocytic Trafficking and Degradation in Pancreatic Beta Cells DOI Open Access

Zijian Fang,

Shiqian Chen,

Yusman Manchanda

et al.

International Journal of Molecular Sciences, Journal Year: 2020, Volume and Issue: 21(21), P. 8404 - 8404

Published: Nov. 9, 2020

The glucagon-like peptide-1 receptor (GLP-1R) is an important regulator of blood glucose homeostasis. Ligand-specific differences in membrane trafficking the GLP-1R influence its signalling properties and therapeutic potential type 2 diabetes. Here, we have evaluated how different factors combine to control post-endocytic recycling versus degradative pathways. Experiments were performed primary islet cells, INS-1 832/3 clonal beta cells HEK293 using biorthogonal labelling determine localisation degradation after treatment with GLP-1, exendin-4 several further agonist peptides. We also characterised effect a rare GLP1R coding variant, T149M, role endosomal peptidase endothelin-converting enzyme-1 (ECE-1), trafficking. Our data reveal GLP-1 associated preferential targeting towards pathway. but not exendin-4, substrate for ECE-1, resultant propensity intra-endosomal degradation, conjunction binding affinity, contributes alterations behaviours degradation. T149M variant shows reduced internalisation responses, which likely be due disruption cytoplasmic region that couples intracellular effectors. These observations provide insights into ligand- genotype-specific can

Language: Английский

Citations

41
Sulfonated red and far-red rhodamines to visualize SNAP- and Halo-tagged cell surface proteins DOI Creative Commons

Ramona Birke,

Julia Ast, Dorien A. Roosen

et al.

Organic & Biomolecular Chemistry, Journal Year: 2022, Volume and Issue: 20(30), P. 5967 - 5980

Published: Jan. 1, 2022

Sulfonated rhodamine dyes allow SNAP- and Halo-tag labelling of cell surface protein fusions. A far-red version can be used for STED nanoscopy.

Language: Английский

Citations

27
De Novo Design of A Membrane‐Anchored Probe for Multidimensional Quantification of Endocytic Dynamics DOI
Kangqiang Qiu,

Ryo Seino,

Guan‐Qun Han

et al.

Advanced Healthcare Materials, Journal Year: 2022, Volume and Issue: 11(8)

Published: Jan. 15, 2022

As a process of cellular uptake, endocytosis, with gradient acidity in different endocytic vesicles, is vital for the homeostasis intracellular nutrients and other functions. To study dynamics pathway, membrane-anchored pH probe, ECGreen, synthesized to visualize vesicles under structured illumination microscopy (SIM), super-resolution technology. Being sensitive increasing fluorescence at low pH, ECGreen can differentiate early late endosomes as well endolysosomes. Meanwhile, membrane anchoring not only improves durability but also provides an excellent anti-photobleaching property long-time imaging SIM. Moreover, by taking these advantages multidimensional analysis model containing spatial, temporal, information successfully developed elucidating their interactions mitochondria during autophagy, reveals fast conversion near plasma membrane.

Language: Английский

Citations

23
Distinct beta-arrestin coupling and intracellular trafficking of metabotropic glutamate receptor homo- and heterodimers DOI Creative Commons
Joon Lee, Alberto J. Gonzalez-Hernandez, Melanie Kristt

et al.

Science Advances, Journal Year: 2023, Volume and Issue: 9(49)

Published: Dec. 6, 2023

The metabotropic glutamate receptors (mGluRs) are family C, dimeric G protein–coupled (GPCRs), which play critical roles in synaptic transmission. Despite an increasing appreciation of the molecular diversity this family, how distinct mGluR subtypes regulated remains poorly understood. We reveal that different group II/III show markedly beta-arrestin (β-arr) coupling and endocytic trafficking. While mGluR2 is resistant to internalization mGluR3 shows transient β-arr coupling, enables endocytosis recycling, mGluR8 form stable complexes, leads efficient lysosomal targeting degradation. Using chimeras mutagenesis, we pinpoint carboxyl-terminal domain regions control trafficking, including identification splice variant with impaired internalization. then use a battery high-resolution fluorescence assays find heterodimerization further expands regulation. Together, work provides insight into relationship between GPCR/β-arr complex formation trafficking while revealing intricacy regulation mGluRs.

Language: Английский

Citations

14
Protein Delivery: If Your GFP (or Other Small Protein) Is in the Cytosol, It Will Also Be in the Nucleus DOI
David C. Luther, Taewon Jeon, Ritabrita Goswami

et al.

Bioconjugate Chemistry, Journal Year: 2021, Volume and Issue: 32(5), P. 891 - 896

Published: April 19, 2021

Intracellular protein delivery is a transformative tool for biologics research and medicine. Delivery into the cytosol allows proteins to diffuse throughout cell access subcellular organelles. Inefficient caused by endosomal entrapment often misidentified as cytosolic delivery. This inaccuracy muddles what should be key checkpoint in assessing efficiency. Green fluorescent (GFP) robust cargo small enough passively from nucleus. Fluorescence of GFP nucleus direct readout effective Here, we highlight recent examples literature accurate assessment using fluorescence

Language: Английский

Citations

30