Contrasting roles for actin in the cellular uptake of cell penetrating peptide conjugates

Abstract

The increased need for macromolecular therapeutics, such as peptides, proteins and nucleotides, to reach intracellular targets necessitates more effective delivery vectors and a higher level of understanding of their mechanism of action. Cell penetrating peptides (CPPs) can transport a range of macromolecules into cells, either through direct plasma membrane translocation or endocytosis. All known endocytic pathways involve cell-cortex remodelling, a process shown to be regulated by reorganisation of the actin cytoskeleton. Here using flow cytometry, confocal microscopy and a variety of actin inhibitors we identify how actin disorganisation in different cell types differentially influences the cellular entry of three probes: the CPP octaarginine – Alexa488 conjugate (R8-Alexa488), octaarginine conjugated Enhanced Green Fluorescent Protein (EGFP-R8), and the fluid phase probe dextran. Disrupting actin organisation in A431 skin epithelial cells dramatically increases the uptake of EGFP-R8 and dextran, and contrasts strongly to inhibitory effects observed with transferrin and R8 attached to the fluorophore Alexa488. This demonstrates that uptake of the same CPP can occur via different endocytic processes depending on the conjugated fluorescent entity. Overall this study highlights how cargo influences cell uptake of this peptide and that the actin cytoskeleton may act as a gateway or barrier to endocytosis of drug delivery vectors.

Strategic Trastuzumab Mediated Crosslinking Driving Concomitant HER2 and HER3 Endocytosis and Degradation in Breast Cancer

“…Actin is a critical regulator of endocytosis, plasma membrane organisation and structure (26,35). Other HER2-crosslinking based approaches have suggested a role for actin/macropinocytosis in HER2 internalisation (21) and this combined with our SEM data led us to examine the actin-dependency of crosslinking.…”

“…SKBR3 cells (120,000) were seeded onto glass coverslips and cultured at 37°C, 5% CO 2 for 48 hr. Tz uptake experiments (+/-crosslinking) were then performed in the continuous presence of DMSO diluent control or 5 µM CytD to disrupt the actin cytoskeleton (26). At the end of the uptake experiment (total CytD/vehicle exposure time = 7.5 hr) cells were fixed in 3% PFA for 15 min, washed 3x with PBS then permeabilised in 0.2% Triton X-100 and washed a further 3x in PBS.…”

“…For this we conducted control versus Tz alone versus Tz+SA experiments in the presence and absence of cytochalasin D (CytD). CytD is an actin filament disrupting agent and widely-used inhibitor of macropinocytosis (26). Confocal microscopy data in Figure 5A depicts SKBR3 cells incubated with Tz alone or Tz+SA in the continuous presence of CytD or DMSO diluent control.…”

Specific Aptamer-Based Probe for Analyzing Biomarker MCP Entry Into Singapore Grouper Iridovirus-Infected Host Cells via Clathrin-Mediated Endocytosis

“…As reported previously, cellular uptake characteristics are also associated with the cytoskeleton (Al Soraj et al, 2012;He et al, 2018). For example, cell-penetrating peptides (CPPs) have been used as vectors for the cellular delivery of therapeutic drugs.…”

EJP18 peptide derived from the juxtamembrane domain of epidermal growth factor receptor represents a novel membrane-active cell-penetrating peptide

“…Plasmids pEV eGFP and pEV eGFP-R8 were described previously [24]. To construct plasmid pEV eGFP-EJP18, the vector for the plasmid was PCR-amplified from pEV eGFP with primers TAGGAATTCAAGCTTAAGCT GAGCAATAACTAGC and CCATGTGGTGGTGGTGGTGGTGCATATGTATATCTC, the insert for the plasmid was PCR-amplified from pEV eGFP with primers CCACCACCACCACCACATGGTGAGCAAGGGCG AG and AGCTTAAGCTTGAATTCCTACAGCAGGCGGCGCAGGGTGCGTTTGCGCACAATATGGCGGCG GCGCATAAACAGCTTGTACAGCTCGTCCATGCCGAG.…”