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Defining the role of the actin cytoskeleton in cellular uptake of cell penetrating peptides

He, Lin 2016. Defining the role of the actin cytoskeleton in cellular uptake of cell penetrating peptides. PhD Thesis, Cardiff University.
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Abstract

The increased need for macromolecular therapeutics, such as proteins and nucleotides, to reach intracellular targets asks for more effective delivery vectors and a higher level of understanding of their mechanism of action. Cell Penetrating Peptides (CPPs) have been shown to deliver a range of macromolecules into cells either through direct plasma membrane translocation or by endocytosis. All known endocytic pathways involve cellcortex remodelling, a process shown to be regulated by reorganisation of the actin cytoskeleton. Links between actin remodelling and CPP uptake has been shown but more information is required to determine the extent of this association and how it could influence further research into improving the delivery capacity of these entities. This project, by using the CPP octaarginine (R8) investigated how actin disorganisation influences the cellular entry of this peptide when attached to a model fluorophore Alexa 488 or Enhanced Green Fluorescent Protein (EGFP). A confocal microscopy technique was initially developed, allowing for high-resolution and spatial characterization of the actin cytoskeleton at different cell depths. Analysis using this developed method was used to highlight that serum starvation has a strong influence on the capacity of R8 to cause membrane blebs and possibly macropinocytosis. Using a range of direct or indirect actin inhibitors this work also highlighted how they can rapidly cause dramatic cellular deformities beyond the level of actin or more subtly affect actin organisation. Further confocal studies revealed that choice of cell line significantly affects the effect of actin disruption on CPP entry and that this is highly dependent on the nature of the probe. This was exemplified by results showing inhibition of EGFP-R8 uptake in HeLa cells treated with cytochalasin D, ! ! ii! latrunculin B and jasplakinolide but a dramatic increase in uptake in A431 cells when they were treated with these drugs. The regulation of actin dynamics involves various kinases including Rho–associated kinases ROCKs, and Src family kinases. The ROCK inhibitor Y27632 induced the formation of actin needles running perpendicular to the plasma membrane of A431 cells and increased EGFP-R8 internalisation. By contrast, Src inhibitor PP2 had little effect on both the actin cytoskeleton and EGFP-R8 uptake but completely abrogated the effects of Cyt D on cellular uptake. This demonstrates for the first time that pretreatment of actin with one inhibitor can negate the endocytic effects of another actin inhibitor working on a different target. Overall this study highlights the importance of analysing actin in detail to identify how CPPs and possibly other drug delivery vectors and formulations interact with cells to gain entry. Under defined experimental conditions R8 can modify the actin cytoskeleton and requires a functional or dysfunctional actin network to allow for maximal cellular entry.

Item Type: Thesis (PhD)
Date Type: Publication
Status: Unpublished
Schools: Pharmacy
Subjects: R Medicine > RM Therapeutics. Pharmacology
Uncontrolled Keywords: Cell penetrating; C ytoskeleton
Date of First Compliant Deposit: 27 September 2016
Last Modified: 07 Oct 2017 01:30
URI: http://orca.cf.ac.uk/id/eprint/94910

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