Imaging Select Mammalian Organelles Using Fluorescent Microscopy: Application to Drug Delivery

Dyer, Paul D.R.; Kotha, Arun K.; Pettit, Marie W.; Richardson, Simon C. W.

doi:10.1007/978-1-62703-336-7_19

Cited by 5 publications

(13 citation statements)

References 9 publications

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“…This "pulse" of GST-GFP was added to complete cell culture media and incubated for 4 hours at 37 o C. The cells were then washed three times with fresh sterile PBS and returned to incubate in complete media for an additional 20h at 37 o C in 5% (v/v) CO2. The cells were then washed 3 times in PBS and fixed with either cold methanol or formaldehyde (as noted) and as previously described (Dyer et al, 2013;Dyer et al, 2015). Immunostaining was performed as previously described (Richardson et al, 2004;Richardson et al, 2008;Dyer et al, 2013;Dyer et al, 2015) using EEA1 (BD Bioscience,) LAMP1 or LAMP2 specific monoclonal antibodies (DHSB hybridoma bank, University of Iowa, USA) as previously described (Richardson et al, 2004;Richardson et al, 2008;Dyer et al, 2015).…”

Section: Methodsmentioning

confidence: 99%

“…EEA1 has been well documented as a Rab5 binding partner (Simonsen et al, 1998), which interacts with membrane inositol phosphates (Simonsen et al, 1998). (Dyer et al, 2013;Richardson et al, 2008;. Figure 2 also shows the effect of probing cells expressing eGFP-Rab5a (panel d) with TxR-WGA (panels e).…”

Section: Over-expressed Gfp-fusion Proteins As Intracellular Markersmentioning

confidence: 98%

“…The expanded vesicular structures positive for both TxR-WGA and eGFP-Rab5a (panel f) were profound. This phenotype has been associated with very high (~50 µg/mL) concentrations of WGA (Dyer et al, 2013) or the constitutively active, GTP locked Rab5 mutant, Rab5:Q79L (Barbieri et al, 1996). Figure 3 (panel a) shows the emission spectra of GST-GFP over time and there was no readily quantifiable alteration in emission spectra over the 300min documented.…”

Section: Over-expressed Gfp-fusion Proteins As Intracellular Markersmentioning

confidence: 99%

“…Equivalently, monoclonal antibodies may be used to identify molecules that act as compartmental "doorkeepers", responsible for regulating fusion to, and consequently the identity of a specific intracellular compartment (Dyer et al, 2013). This is considerably more time consuming and expensive than generating polyclonal antisera.…”

Section: Syntaxin13 Typically Catalyses Vesicle Fusion Events Betweenmentioning

confidence: 99%

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Green fluorescent protein (GFP): is seeing believing and is that enough?

Shorter

Pettit

Dyer

et al. 2017

Journal of Drug Targeting

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Intracellular compartmentalisation is a significant barrier to the successful nucleocytosolic delivery of biologics. The endocytic system has been shown to be responsible for compartmentalisation, providing an entry point, and trigger(s) for the activation of drug delivery systems. Consequently, many of the technologies used to understand endocytosis have found utility within the field of drug delivery. The use of fluorescent proteins as markers denoting compartmentalisation within the endocytic system has become commonplace. Several of the limitations associated with the use of green fluorescent protein (GFP) within the context of drug delivery have been explored here by asking a series of related questions: (1) Are molecules that regulate fusion to a specific compartment (i.e. Rab- or SNARE-GFP fusions) a good choice of marker for that compartment? (2) How reliable was GFP-marker overexpression when used to define a given endocytic compartment? (3) Can glutathione-s-transferase (GST) fused in frame with GFP (GST-GFP) act as a fluid phase endocytic probe? (4) Was GFP fluorescence a robust indicator of (GFP) protein integrity? This study concluded that there are many appropriate and useful applications for GFP; however, thought and an understanding of the biological and physicochemical character of these markers are required for the generation of meaningful data.

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Section: Methodsmentioning

confidence: 99%

Section: Over-expressed Gfp-fusion Proteins As Intracellular Markersmentioning

confidence: 98%

Section: Over-expressed Gfp-fusion Proteins As Intracellular Markersmentioning

confidence: 99%

Section: Syntaxin13 Typically Catalyses Vesicle Fusion Events Betweenmentioning

confidence: 99%

See 2 more Smart Citations

Green fluorescent protein (GFP): is seeing believing and is that enough?

Shorter

Pettit

Dyer

et al. 2017

Journal of Drug Targeting

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“…Co‐localization between the parasite and EEA1‐positive structures was investigated, with axenic amastigotes co‐localizing at 10 min and up to 4 h after infection. Co‐localization with EEA‐1‐positive compartments often occurs within 5–10 min of entry via the endocytic system, prior to translocation to other subcellular organelles (e.g., late endosome, Golgi apparatus) within 1 h . The prolonged retention of axenic amastigotes within EEA‐1‐positive structures suggests a mechanism of interaction with the endosomal sorting machinery.…”

Section: Introductionmentioning

confidence: 99%

Leishmania aethiopica cell‐to‐cell spreading involves caspase‐3, AkT, and NF‐κB but not PKC‐δ activation and involves uptake of LAMP‐1‐positive bodies containing parasites

et al. 2019

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Development of human leishmaniasis is dependent on the ability of intracellular Leishmania parasites to spread and enter macrophages. The mechanism through which free promastigotes and amastigotes bind and enter host macrophages has been previously investigated; however, little is known about intracellular trafficking and cell-to-cell spreading. In this study, the mechanism involved in the spreading of Leishmania aethiopica and Leishmania mexicana was investigated. A significant increase in phosphatidylserine (PS) exhibition, cytochrome C release, and active caspase-3 expression was detected (P < 0.05) during L. aethiopica, but not L. mexicana spreading. A decrease (P < 0.05) of protein kinase B (Akt) protein and BCL2-associated agonist of cell death (BAD) phosphorylation was also observed. The nuclear factor kappa-lightchain enhancer of activated B cells (NF-kB) signaling pathway and pro-apoptotic protein protein kinase C delta (PKC-d) were downregulated while inhibition of caspase-3 activation prevented L. aethiopica spreading. Overall suggesting that L. aethiopica induces host cell's apoptosis during spreading in a caspase-3-dependent manner. The trafficking of amastigotes within macrophages following cell-to-cell spreading differed from that of axenic parasites and involved co-localization with lysosomal-associated membrane protein 1 (LAMP-1) within 10 min postinfection. Interestingly, following infection with axenic amastigotes and promastigotes, co-localization of parasites with LAMP-1-positive structures took place at 1 and 4 h, respectively, suggesting that the membrane coat and LAMP-1 protein were derived from the donor cell. Collectively, these findings indicate that host cell apoptosis, demonstrated by PS exhibition, caspase-3 activation, cytochrome C release, downregulation of Akt, BAD phosphorylation, NF-kB activation, and independent of PKC-d expression, is involved in L. aethiopica spreading. Moreover, L. aethiopica parasites associate with LAMP-rich structures when taken up by neighboring macrophages.Abbreviations 7AAD, 7-aminoactinomycin D; Akt, protein kinase B; BAD, BCL2-associated agonist of cell death; CF, PKC-d cleaved fragment; CL, cutaneous leishmaniasis; EEA 1, early endosome antigen 1; HI-FBS, heat-inactivated fetal bovine serum; IF1, intermediate form 1; LAMP-1, lysosomal-associated membrane protein 1; NF-kB, nuclear factor kappa-light-chain enhancer of activated B cells; PKC-d, protein kinase C delta; PMA, phorbol 12-myristate 13-acetate; PS, phosphatidylserine; RA, retinoic acid; SEM, scanning electron microscope; Ser, serine; Thr, threonine.

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Disarmed anthrax toxin delivers antisense oligonucleotides and siRNA with high efficiency and low toxicity

Dyer

Shepherd

Gollings

et al. 2015

Journal of Controlled Release

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Inefficient cytosolic delivery and vector toxicity contribute to the limited use of antisense oligonucleotides (ASOs) and siRNA as therapeutics. As anthrax toxin (Atx) accesses the cytosol, the purpose of this study was to evaluate the potential of disarmed Atx to deliver either ASOs or siRNA. We hypothesized that this delivery strategy would facilitate improved transfection efficiency while eliminating the toxicity seen for many vectors due to membrane destabilization. Atx complex formation with ASOs or siRNA was achieved via the in-frame fusion of either Saccharomyces cerevisiae GAL4 or Homo sapien sapien PKR (respectively) to a truncation of Atx lethal factor (LFn), which were used with Atx protective antigen (PA). Western immunoblotting confirmed the production of: LFN-GAL4, LFn-PKR and PA which were detected at ~45.9 kDa, ~37 kDa, and ~83 kDa respectively and small angle neutron scattering confirmed the ability of PA to form an annular structure with a radius of gyration of 7.0 ± 1.0 nm when placed in serum. In order to form a complex with LFn-GAL4, ASOs were engineered to contain a double-stranded region, and a cell free in vitro translation assay demonstrated that no loss of antisense activity above 30 pmol ASO was evident. The in vitro toxicity of both PA:LFn-GAL4:ASO and PA:LFn-PKR:siRNA complexes was low (IC50>100 μg/mL in HeLa and Vero cells) and subcellular fractionation in conjunction with microscopy confirmed the detection of LFn-GAL4 or LFn-PKR in the cytosol. Syntaxin5 (Synt5) was used as a model target gene to determine pharmacological activity. The PA:LFn-GAL4:ASO complexes had transfection efficiency approximately equivalent to Nucleofection® over a variety of ASO concentrations (24h post-transfection) and during a 72 h time course. In HeLa cells, at 200 pmol ASO (with PA:LFN-GAL4), 5.4 ± 2.0% Synt5 expression was evident relative to an untreated control after 24h. Using 200 pmol ASOs, Nucleofection® reduced Synt5 expression to 8.1 ± 2.1% after 24h. PA:LFn-GAL4:ASO transfection of non- or terminally-differentiated THP-1 cells and Vero cells resulted in 35.2 ± 19.1%, 36.4 ± 1.8% and 22.9 ± 6.9% (respectively) Synt5 expression after treatment with 200 pmol of ASO and demonstrated versatility. Nucleofection® with Stealth RNAi™ siRNA reduced HeLa Synt5 levels to 4.6 ± 6.1% whereas treatment with the PA:LFn-PKR:siRNA resulted in 8.5 ± 3.4% Synt5 expression after 24h (HeLa cells). These studies report for the first time an ASO and RNAi delivery system based upon protein toxin architecture that is devoid of polycations. This system may utilize regulated membrane back-fusion for the cytosolic delivery of ASOs and siRNA, which would account for the lack of toxicity observed. High delivery efficiency suggests further in vivo evaluation is warranted.

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Imaging Select Mammalian Organelles Using Fluorescent Microscopy: Application to Drug Delivery

Cited by 5 publications

References 9 publications

Green fluorescent protein (GFP): is seeing believing and is that enough?

Green fluorescent protein (GFP): is seeing believing and is that enough?

Leishmania aethiopica cell‐to‐cell spreading involves caspase‐3, AkT, and NF‐κB but not PKC‐δ activation and involves uptake of LAMP‐1‐positive bodies containing parasites

Disarmed anthrax toxin delivers antisense oligonucleotides and siRNA with high efficiency and low toxicity

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