Tracking Intracellular Polymer Localization Via Fluorescence Microscopy

Richardson, Simon C. W.

doi:10.1002/9780470875780.ch10

Cited by 3 publications

(5 citation statements)

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“…Membrane and vesicle shedding is also important in cell–cell communication, e.g., via exosomes and trogocytosis. The fluorescent images depict Vero cells stained using specific organelle markers (for methods see refs , , and ). Table gives more details of the constituent organelles and the markers used to identify them.…”

Section: Endocytosis and Intracellular Traffickingmentioning

confidence: 99%

“…Although intracellular vesicles are often labeled as endosomes or LYS based simply on morphological appearance in a TEM or fluorescence microscopy images, intracellular organelle positioning and morphology are often very different in each cell type, making this kind of assumption unhelpful. It is important to identify subcellular compartments using “landmarks” or markers (reviewed in ref ). As internalized receptors, cargos, and indeed many of the regulatory proteins only transiently associate with a specific compartment (there may be latent protein pools elsewhere in the cell, e.g., the EEA1 cytosolic pool), there is a continuing search for reliable organelle markers applicable to all cell types.…”

Section: Endocytosis and Intracellular Traffickingmentioning

confidence: 99%

“…Colocalization techniques offer the possibility of organelle identification, and such studies usually use one or more of the following approaches: (i) a pulse-chase experiment using exogenously applied marker substrates, e.g., Transferrin-Texas Red (to mark EE), fluorescein isothiocyanate (FITC)-dextran (to mark LYS) etc., (ii) cells transfected with green fluorescence protein (GFP)-labeled organelle marker proteins, or (iii) following cell fixation use of labeled antibodies against the organelle marker proteins (Table ). Protocols for, and the pros and cons of, fluorescence microscopy techniques are discussed at length elsewhere. ,,, Briefly, the main issues are as follows: these are single cell assays (are the results typical of the whole population? ); lack of quantitation; difficulty in obtaining a temporal dissection of the trafficking pathway; and, where fixation is used, the potential for fixation artifacts.…”

Section: Nanomedicine Endocytosis: Quantitationmentioning

confidence: 99%

“…Protocols for, and the pros and cons of, fluorescence microscopy techniques are discussed at length elsewhere. 41,50,54,55 Briefly, the main issues are as follows: these are single cell assays (are the results typical of the whole population? ); lack of quantitation; difficulty in obtaining a temporal dissection of the trafficking pathway; and, where fixation is used, the potential for fixation artifacts.…”

Section: Molecular Pharmaceuticsmentioning

confidence: 99%

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Endocytosis and Intracellular Trafficking as Gateways for Nanomedicine Delivery: Opportunities and Challenges

2012

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More than 40 nanomedicines are already in routine clinical use with a growing number following in preclinical and clinical development. The therapeutic objectives are often enhanced disease-specific targeting (with simultaneously reduced access to sites of toxicity) and, especially in the case of macromolecular biotech drugs, improving access to intracellular pharmacological target receptors. Successful navigation of the endocytic pathways is usually a prerequisite to achieve these goals. Thus a comprehensive understanding of endocytosis and intracellular trafficking pathways in both the target and bystander normal cell type(s) is essential to enable optimal nanomedicine design. It is becoming evident that endocytic pathways can become disregulated in disease and this, together with the potential changes induced during exposure to the nanocarrier itself, has the potential to significantly impact nanomedicine performance in terms of safety and efficacy. Here we overview the endomembrane trafficking pathways, discuss the methods used to determine and quantitate the intracellular fate of nanomedicines, and review the current status of lysosomotropic and endosomotropic delivery. Based on the lessons learned during more than 3 decades of clinical development, the need to use endocytosis-relevant clinical biomarkers to better select those patients most likely to benefit from nanomedicine therapy is also discussed.

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Section: Endocytosis and Intracellular Traffickingmentioning

confidence: 99%

Section: Endocytosis and Intracellular Traffickingmentioning

confidence: 99%

Section: Nanomedicine Endocytosis: Quantitationmentioning

confidence: 99%

Section: Molecular Pharmaceuticsmentioning

confidence: 99%

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Endocytosis and Intracellular Trafficking as Gateways for Nanomedicine Delivery: Opportunities and Challenges

2012

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“…Most intracellular markers occupy more than one intracellular compartment, a specific example being the transferrin receptor, which is often used as a marker for recycling endosomes (i.e. a Rab11 enriched compartment) (Richardson, 2010). This receptor cycles through early and recycling endosomes en route to the plasma membrane, and back to the early endosome after internalisation.…”

Section: Discussionmentioning

confidence: 99%

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

Dyer

Kotha

Pettit

et al. 2013

Methods in Molecular Biology

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The microscopic imaging of specific organelles has become a staple of the single-cell assay and has helped define the molecular regulation of many physiological processes. This definition has been made possible by utilizing different criteria to identify specific subpopulations of organelles. These criteria can be biochemical, immunological, or physiological, and in many cases, markers regulate fusion to the organelle they define (e.g., Rab-GTPase proteins). Single-cell imaging technology allows, within the context of drug delivery, an evaluation of the intracellular trafficking of both biological and synthetic macromolecules. However, it should be remembered that there are many limitations associated with this type of study and quantitation is not easy. The temporal dissection of novel and default trafficking of both macromolecular "drugs" and macromolecular drug delivery systems is possible. These methodologies are detailed herein.

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Tracking Intracellular Polymer Localization Via Fluorescence Microscopy

Cited by 3 publications

References 53 publications

Endocytosis and Intracellular Trafficking as Gateways for Nanomedicine Delivery: Opportunities and Challenges

Endocytosis and Intracellular Trafficking as Gateways for Nanomedicine Delivery: Opportunities and Challenges

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

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

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