Super-resolution microscopy unveils transmembrane domain-mediated internalization of cross-reacting material 197 into diphtheria toxin-resistant mouse J774A.1 cells and primary rat fibroblasts in vitro
Abstract:Diphtheria toxin (DT) efficiently inhibits protein synthesis in human cells, resulting in severe disease diphtheria. The sensitivity towards DT varies between mammalian species. Mice and rats are resistant to DT. However, the reason underlying this insensitivity is controversially discussed and not well understood. Therefore, we investigated the steps of DT uptake, i.e. receptor binding and internalization into mouse J774A.1 macrophages and primary rat fibroblasts. We exploited the nontoxic DT-mutant cross-rea… Show more
“…Initially, the different cells were treated separately with His_ eGFP_C3bot E174Q , and internalization of eGFP was quantified in flow cytometry ( Figure 2 a). Since only internalized cargo should be analyzed, cell-surface-bound eGFP signals were quenched by using an established trypan-blue-based assay [ 38 , 39 , 40 ]. Uptake of His_ eGFP_C3bot E174Q into macrophages was strongly enhanced in comparison with lymphocytes, which internalized only a minor portion of the fusion protein ( Figure 2 a).…”
Section: Resultsmentioning
confidence: 99%
“…Afterwards, cells were washed with FACS buffer (1% FCS (Biochrom, Berlin, Germany) and 0.1% sodium azide (VWR, Radnor, PA, USA) in PBS (Gibco-Life Technologies, Carlsbad, CA, USA) and centrifuged for 10 min at 1300 rpm. The supernatant was discarded, and cells were incubated directly before measurement for 1 min with 50 µg/mL trypan blue to quench extracellular eGFP signals as described in [ 40 ]. Intracellular fluorescence was detected using a FACSCalibur™ flow cytometer (BD Biosciences, Heidelberg, Germany) .…”
The protein toxin C3bot from Clostridium botulinum is a mono-ADP-ribosyltransferase that selectively intoxicates monocyte-derived cells such as macrophages, osteoclasts, and dendritic cells (DCs) by cytosolic modification of Rho-A, -B, and -C. Here, we investigated the application of C3bot as well as its non-toxic variant C3botE174Q as transporters for selective delivery of cargo molecules into macrophages and DCs. C3bot and C3botE174Q facilitated the uptake of eGFP into early endosomes of human-monocyte-derived macrophages, as revealed by stimulated emission depletion (STED) super-resolution microscopy. The fusion of the cargo model peptide eGFP neither affected the cell-type selectivity (enhanced uptake into human macrophages ex vivo compared to lymphocytes) nor the cytosolic release of C3bot. Moreover, by cell fractionation, we demonstrated that C3bot and C3botE174Q strongly enhanced the cytosolic release of functional eGFP. Subsequently, a modular system was created on the basis of C3botE174Q for covalent linkage of cargos via thiol–maleimide click chemistry. The functionality of this system was proven by loading small molecule fluorophores or an established reporter enzyme and investigating the cellular uptake and cytosolic release of cargo. Taken together, non-toxic C3botE174Q is a promising candidate for the cell-type-selective delivery of small molecules, peptides, and proteins into the cytosol of macrophages and DCs.
“…Initially, the different cells were treated separately with His_ eGFP_C3bot E174Q , and internalization of eGFP was quantified in flow cytometry ( Figure 2 a). Since only internalized cargo should be analyzed, cell-surface-bound eGFP signals were quenched by using an established trypan-blue-based assay [ 38 , 39 , 40 ]. Uptake of His_ eGFP_C3bot E174Q into macrophages was strongly enhanced in comparison with lymphocytes, which internalized only a minor portion of the fusion protein ( Figure 2 a).…”
Section: Resultsmentioning
confidence: 99%
“…Afterwards, cells were washed with FACS buffer (1% FCS (Biochrom, Berlin, Germany) and 0.1% sodium azide (VWR, Radnor, PA, USA) in PBS (Gibco-Life Technologies, Carlsbad, CA, USA) and centrifuged for 10 min at 1300 rpm. The supernatant was discarded, and cells were incubated directly before measurement for 1 min with 50 µg/mL trypan blue to quench extracellular eGFP signals as described in [ 40 ]. Intracellular fluorescence was detected using a FACSCalibur™ flow cytometer (BD Biosciences, Heidelberg, Germany) .…”
The protein toxin C3bot from Clostridium botulinum is a mono-ADP-ribosyltransferase that selectively intoxicates monocyte-derived cells such as macrophages, osteoclasts, and dendritic cells (DCs) by cytosolic modification of Rho-A, -B, and -C. Here, we investigated the application of C3bot as well as its non-toxic variant C3botE174Q as transporters for selective delivery of cargo molecules into macrophages and DCs. C3bot and C3botE174Q facilitated the uptake of eGFP into early endosomes of human-monocyte-derived macrophages, as revealed by stimulated emission depletion (STED) super-resolution microscopy. The fusion of the cargo model peptide eGFP neither affected the cell-type selectivity (enhanced uptake into human macrophages ex vivo compared to lymphocytes) nor the cytosolic release of C3bot. Moreover, by cell fractionation, we demonstrated that C3bot and C3botE174Q strongly enhanced the cytosolic release of functional eGFP. Subsequently, a modular system was created on the basis of C3botE174Q for covalent linkage of cargos via thiol–maleimide click chemistry. The functionality of this system was proven by loading small molecule fluorophores or an established reporter enzyme and investigating the cellular uptake and cytosolic release of cargo. Taken together, non-toxic C3botE174Q is a promising candidate for the cell-type-selective delivery of small molecules, peptides, and proteins into the cytosol of macrophages and DCs.
“…Both the EEA1 and LL-37 form donut shape structures, indicating that both are localized in the endosomal membrane. The donut-like shape is unlikely to be an artifact, since molecules that are located inside endosomal vesicles appear as punctate objects inside the lumen of the endosomes in STED images [ 42 ]. To check the successful internalization of Mtb in macrophages under our experimental conditions, we performed a LAM-staining on intracellular Mtb and a microtubule staining for the macrophages ( Supplementary Figure S4 ).…”
The antimicrobial peptide LL-37 inhibits the growth of the major human pathogen Mycobacterium tuberculosis (Mtb), but the mechanism of the peptide–pathogen interaction inside human macrophages remains unclear. Super-resolution imaging techniques provide a novel opportunity to visualize these interactions on a molecular level. Here, we adapt the super-resolution technique of stimulated emission depletion (STED) microscopy to study the uptake, intracellular localization and interaction of LL-37 with macrophages and virulent Mtb. We demonstrate that LL-37 is internalized by both uninfected and Mtb infected primary human macrophages. The peptide localizes in the membrane of early endosomes and lysosomes, the compartment in which mycobacteria reside. Functionally, LL-37 disrupts the cell wall of intra- and extracellular Mtb, resulting in the killing of the pathogen. In conclusion, we introduce STED microscopy as an innovative and informative tool for studying host–pathogen–peptide interactions, clearly extending the possibilities of conventional confocal microscopy.
“…Moreover, we conjugated SP9–Cy3 with biotin and bound it to streptavidin-conjugated bacterial toxins (non-toxic mutants of clostridial C2 toxin or diphtheria toxin (CRM197)) as a possible alternative for intracellular delivery. These toxins can deliver biotin-conjugated peptides into mammalian cells through endocytosis 46 , 47 . Fig.…”
Section: Sp9 Inhibits Stimulated Mucin Secretion In Cellsmentioning
Membrane fusion triggered by Ca2+ is orchestrated by a conserved set of proteins to mediate synaptic neurotransmitter release, mucin secretion and other regulated exocytic processes1–4. For neurotransmitter release, the Ca2+ sensitivity is introduced by interactions between the Ca2+ sensor synaptotagmin and the SNARE complex5, and sequence conservation and functional studies suggest that this mechanism is also conserved for mucin secretion6. Disruption of Ca2+-triggered membrane fusion by a pharmacological agent would have therapeutic value for mucus hypersecretion as it is the major cause of airway obstruction in the pathophysiology of respiratory viral infection, asthma, chronic obstructive pulmonary disease and cystic fibrosis7–11. Here we designed a hydrocarbon-stapled peptide that specifically disrupts Ca2+-triggered membrane fusion by interfering with the so-called primary interface between the neuronal SNARE complex and the Ca2+-binding C2B domain of synaptotagmin-1. In reconstituted systems with these neuronal synaptic proteins or with their airway homologues syntaxin-3, SNAP-23, VAMP8, synaptotagmin-2, along with Munc13-2 and Munc18-2, the stapled peptide strongly suppressed Ca2+-triggered fusion at physiological Ca2+ concentrations. Conjugation of cell-penetrating peptides to the stapled peptide resulted in efficient delivery into cultured human airway epithelial cells and mouse airway epithelium, where it markedly and specifically reduced stimulated mucin secretion in both systems, and substantially attenuated mucus occlusion of mouse airways. Taken together, peptides that disrupt Ca2+-triggered membrane fusion may enable the therapeutic modulation of mucin secretory pathways.
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