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Fluorescent membrane markers elucidate the association of Borrelia burgdorferi with tick cell lines
摘要： This study aimed to describe the association of Borrelia burgdorferi s.s. with ixodid tick cell lines by flow cytometry and fluorescence and confocal microscopy. Spirochetes were stained with a fluorescent membrane marker (PKH67 or PKH26), inoculated into 8 different tick cell lines and incubated at 30°C for 24 h. PKH efficiently stained B. burgdorferi without affecting bacterial viability or motility. Among the tick cell lines tested, the Rhipicephalus appendiculatus cell line RA243 achieved the highest percentage of association/internalization, with both high (90%) and low (10%) concentrations of BSK-H medium in tick cell culture medium. Treatment with cytochalasin D dramatically reduced the average percentage of cells with internalized spirochetes, which passed through a dramatic morphological change during their internalization by the host cell as observed in time-lapse photography. Almost all of the fluorescent bacteria were seen to be inside the tick cells. PKH labeling of borreliae proved to be a reliable and valuable tool to analyze the association of spirochetes with host cells by flow cytometry, confocal and fluorescence microscopy.
关键词： Fluorescent membrane marker,Tick cell lines,Borrelia burgdorferi,Phagocytosis
更新于2025-11-21 11:24:58
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A Photostable AIEgen for Specific and Real-time Monitoring of Lysosomal Processes
摘要： Lysosomes are recognized as advanced organelles involved in many cellular processes, and are also considered as crucial regulators of cell homeostasis. The current strategies for monitoring activities of lysosomes exhibit some limitations. Herein, we synthesized a novel fluorescent probe named 2M-DPAS with AIE characteristics, which was proved to have significant advantages of good biocompatibility, high selectivity, bright emission and excellent photostability. Based on those, 2M-DPAS can be used to continuously monitor the dynamic changes of lysosomes including autophagy and mitophagy, as well as to track the process of endocytosis of macromolecules in lysosomes, which are benefit to better know about the lysosomes-related diseases.
关键词： Aggregation-induced emission,Lysosomes,Autophagy,Monitoring,Phagocytosis of macromolecules
更新于2025-09-23 15:23:52
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Early Onset Ultrastructural and Functional Defects in RPE and Photoreceptors of a Stargardt-Like Macular Dystrophy (STGD3) Transgenic Mouse Model
摘要： PURPOSE. We investigated the interplay between photoreceptors expressing mutant ELOVL4 (responsible for Stargardt-like disease, STGD3) and RPE in the initial stages of retinal degeneration. METHODS. Using electron microscopy and electroretinogram (ERG), we assessed RPE and photoreceptor ultrastructure and function in transgenic ELOVL4 (TG1-2 line; TG) and wild-type (WT) littermates. Experiments were done at P30, 1 month before photoreceptor loss in TG and at P90, a time point with approximately 30% rod loss. To further elucidate the mechanism underlying our ultrastructural and functional results, we undertook Western blotting and immunohistochemistry of key proteins involved in phagocytosis of outer segments by RPE cells. RESULTS. Firstly, we showed that in TG mouse photoreceptors, endogenous ELOVL4 protein is not mislocalized in the presence of the mutated ELOVL4 protein. Secondly, we found evidence of RPE toxicity at P30, preceding any photoreceptor loss. Pathology in RPE cells was exacerbated at P90. Furthermore, higher proportions of phagosomes remained at the apical side of RPE cells. Subretinal lysosomal deposits were immunopositive for phagocytic proteins. Ultrastructural analysis of photoreceptor (rod) outer segments showed disrupted surface morphology consisting of disc spacing irregularities. Finally, rods and RPE exhibited signs of dysfunction as measured by the ERG a-wave leading edge (P30) and c-wave (P90), respectively. CONCLUSIONS. The presence of human mutant ELOVL4 in transgenic mouse photoreceptors leads to early outer segment disc pathology and RPE cytotoxicity. Defective processing of these abnormal discs by RPE cells ultimately may be responsible for outer segment truncation, photoreceptor death, and vision loss.
关键词： mouse,outer segment,Stargardt-like dystrophy,photoreceptor,STGD3,phagocytosis,ELOVL4,RPE
更新于2025-09-23 15:23:52
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[Methods in Molecular Biology] Autophagy Volume 1880 (Methods and Protocols) || Correlative Light and Electron Microscopy to Analyze LC3 Proteins in Caenorhabditis elegans Embryo
摘要： In this chapter, we present a protocol to perform correlative light and electron microscopy (CLEM) on Caenorhabditis elegans embryos. We use a specific fixation method which preserves both the GFP fluorescence and the structural integrity of the samples. Thin sections are first analyzed by light microscopy to detect GFP-tagged proteins, then by transmission electron microscopy (TEM) to characterize the ultrastructural anatomy of cells. The superimposition of light and electron images allows to determine the subcellular localization of the fluorescent protein. We have used this method to characterize the roles of autophagy in the phagocytosis of apoptotic cells in C. elegans embryos. We analyzed in apoptotic cell and phagocytic cell the localization of the two homologs of LC3/GABARAP proteins, namely, LGG-1 and LGG-2.
关键词： LC3-associated phagocytosis,High-pressure freezing,Freeze substitution,LGG-2,Green fluorescent protein,GMA resin,LGG-1
更新于2025-09-23 15:22:29
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A Fluorescence Based-Proliferation Assay for the Identification of Replicating Bacteria Within Host Cells
摘要： Understanding host pathogen interactions is paramount to the development of novel antimicrobials. An important facet of this pursuit is the accurate characterization of pathogen replication within infected host cells. Here we describe the use of a fluorescence-based proliferation assay to identify intracellular populations of replicating bacteria at the subcellular level. Using Staphylococcus aureus as a model Gram-positive bacterial pathogen and macrophages as a model host phagocyte, we demonstrate this assay can be used to reliably identify individual phagocytes that contain replicating bacteria. Furthermore, we demonstrate this assay is compatible with additional cellular probes that enable characterization of cellular compartments in which replicating bacteria reside. Finally, we demonstrate that this assay facilitates the investigation of both Gram-negative and Gram-positive bacteria within host cells.
关键词： microscopy,Staphylococcus,phagolysosome,fluorescence,phagocytosis,macrophage
更新于2025-09-23 15:22:29
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Bisretinoids mediate light sensitivity resulting in photoreceptor cell degeneration in mice lacking the receptor tyrosine kinase Mer
摘要： The receptor tyrosine kinase Mer is expressed by retinal pigment epithelial (RPE) cells and participates in photoreceptor outer-segment phagocytosis, a process enabling membrane renewal. Mutations in the gene encoding MERTK cause blinding retinitis pigmentosa in humans. Targeted Mertk disruption in mice causes defective RPE-mediated phagocytosis of the outer segments, leading to deposition of autofluorescent debris at the RPE–photoreceptor cell interface, followed by photoreceptor cell degeneration. Here, we show that retinaldehyde adducts (bisretinoid fluorophores) that form in photoreceptor outer segments occupy the unphagocytosed outer segment debris that accumulates in Mertk-/- mice. Bisretinoids measured by HPLC were elevated in Mertk-/- mice compared with wild-type animals. Bisretinoids were also more abundant in albino Mertk-/- mice expressing leucine at position 450 of the isomerase RPE65 (Rpe65-Leu450) rather than the variant methionine (Rpe65-450Met) that yields lower bisretinoid levels. In Royal College of Surgeons rats having dysfunctional Mertk, bisretinoids were higher than in wild-type rats. Intensities of in vivo fundus autofluorescence were higher in Mertk-/- mice than in wild-type mice and peaked earlier in albino Mertk-/-/Rpe65-Leu450 mice than in albino Mertk-/-/Rpe65-450Met mice. Of note, the rate of photoreceptor cell degeneration was more rapid in albino Mertk-/- mice exposed to higher levels of intraocular light (albino versus pigmented mice) and in mice carrying Rpe65-Leu450 than in Rpe65-450Met mice, revealing a link between bisretinoid accumulation and light-mediated acceleration of photoreceptor cell degeneration. In conclusion, the light sensitivity of photoreceptor cell degeneration arising from Mertk deficiency is consistent with the known phototoxicity of bisretinoids.
关键词： Mertk-/-,Mertk,retinal pigment epithelium,lipofuscin,phagocytosis,retina,photodegradation,bisretinoid
更新于2025-09-23 15:21:01
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Kif17 phosphorylation regulates photoreceptor outer segment turnover
摘要： Background: KIF17, a kinesin-2 motor that functions in intraflagellar transport, can regulate the onset of photoreceptor outer segment development. However, the function of KIF17 in a mature photoreceptor remains unclear. Additionally, the ciliary localization of KIF17 is regulated by a C-terminal consensus sequence (KRKK) that is immediately adjacent to a conserved residue (mouse S1029/zebrafish S815) previously shown to be phosphorylated by CaMKII. Yet, whether this phosphorylation can regulate the localization, and thus function, of KIF17 in ciliary photoreceptors remains unknown. Results: Using transgenic expression in zebrafish photoreceptors, we show that phospho-mimetic KIF17 has enhanced localization along the cone outer segment. Importantly, expression of phospho-mimetic KIF17 is associated with greatly enhanced turnover of the photoreceptor outer segment through disc shedding in a cell-autonomous manner, while genetic mutants of kif17 in zebrafish and mice have diminished disc shedding. Lastly, cone expression of constitutively active tCaMKII leads to a kif17-dependent increase in disc shedding. Conclusions: Taken together, our data support a model in which phosphorylation of KIF17 promotes its photoreceptor outer segment localization and disc shedding, a process essential for photoreceptor maintenance and homeostasis. While disc shedding has been predominantly studied in the context of the mechanisms underlying phagocytosis of outer segments by the retinal pigment epithelium, this work implicates photoreceptor-derived signaling in the underlying mechanisms of disc shedding.
关键词： Disc shedding,Retina,Phagocytosis,Photoreceptor,Cilia,Intraflagellar transport,Kinesin
更新于2025-09-23 15:21:01
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Differences between Neutrophilic Granulocytes and Lymphocytes in Fixation of Quantum Dots of Different Composition
摘要： Studies by flow cytometry, scanning electron microscopy, and X-ray microanalysis have shown that fixation/internalization of quantum dots differ in lymphocytes and neutrophilic granulocytes. Punctate fixation of quantum dots to cell surface is characteristic of lymphocytes, while phagocytosis with perinuclear location of quantum dot aggregations is characteristic of neutrophils. The presence of complex functional groups on the surface of quantum dots can inhibit significantly the fixation/internalization of quantum dots by blood cells.
关键词： lymphocytes,neutrophilic granulocytes,internalization,quantum dots,phagocytosis
更新于2025-09-11 14:15:04
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Müller glia phagocytose dead photoreceptor cells in a mouse model of retinal degenerative disease
摘要： Retinitis pigmentosa is a devastating, blinding disorder that affects 1 in 4000 people worldwide. During the progression of the disorder, phagocytic clearance of dead photoreceptor cell bodies has a protective role by preventing additional retinal damage from accumulation of cellular debris. However, the cells responsible for the clearance remain unidentified. Taking advantage of a mouse model of retinitis pigmentosa (RhoP23H/P23H), we clarified the roles of M ¨uller glia in the phagocytosis of rod photoreceptor cells. During the early stage of retinal degeneration, M ¨uller glial cells participated in the phagocytosis of dying or dead rod photoreceptors throughout the outer nuclear layer. Nearly 50% of M ¨uller glia engaged in phagocytosis. Among the M ¨uller phagosomes, >90% matured into phagolysosomes. Those observations indicated that M ¨uller glial cells are the primary contributor to phagocytosis. In contrast, macrophages migrate to the inner part of the outer nuclear layer during photoreceptor degeneration, participating in the phagocytosis of a limited population of dying or dead photoreceptor cells. In healthy retinas of wild-type mice, M ¨uller glial cells phagocytosed cell bodies of dead rod photoreceptors albeit at a lower frequency. Taken together, the phagocytic function of M ¨uller glia is responsible for retinal homeostasis and reorganization under normal and pathologic conditions.
关键词： retinitis pigmentosa,phagocytosis,rod photoreceptors,rhodopsin
更新于2025-09-10 09:29:36
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Imaging flow cytometry and confocal microscopy-based examination of F-actin and phosphoinositide dynamics during leukocyte immune-type receptor-mediated phagocytic events
摘要： Cells of the innate immune system rapidly detect and eliminate invading microbes using surface-expressed immunoregulatory receptors that translate extracellular binding events into potent effector responses. Channel catfish (Ictalurus punctatus) leukocyte immune-type receptors (IpLITRs) are a family of immunoregulatory proteins that have been shown to regulate several innate immune cell effector responses including the phagocytic process. The mechanisms by which these receptors regulate phagocytosis are not entirely understood but we have previously shown that different IpLITR-types use ITAM-dependent as well as ITAM-independent pathways for controlling target engulfment. The main objective of this study was to develop and use imaging flow cytometry and confocal microscopy-based assays to further examine both F-actin and phosphoinositide dynamics that occur during the different IpLITR-mediated phagocytic pathways. Results show that the ITAM-dependent IpLITR-induced phagocytic response promotes canonical changes in F-actin polymerization and PI(4,5)P2 redistributions. However, the ITAM-independent IpLITR phagocytic response induced unique patterns of F-actin and PI(4,5)P2 redistributions, which are likely due to its ability to regulate alternative signaling pathways. Additionally, both IpLITR-induced phagocytic pathways induced target internalization into PI(3)P-enriched phagosomes indicative of a maturing phagosome compartment. Overall, this imaging-based platform can be further applied to monitor the recruitment and distribution of signaling molecules during IpLITR-mediated phagocytic processes and may serve as a useful strategy for functional examinations of other immunoregulatory receptor-types in fish.
关键词： Cytoskeletal dynamics,Teleost,Phosphoinositides,Leukocyte immune-type receptors,Imaging,Phagocytosis,Innate immunity,Fluorescent probes,F-actin,Flow cytometry
更新于2025-09-04 15:30:14