Abstract
BackgroundThe plasma membrane plays an essential role in selective permeability, compartmentalization, osmotic balance, and cellular uptake. The characteristics and functions of cyanobacterial membranes have been extensively investigated in recent years. Cell-penetrating peptides (CPPs) are special nanocarriers that can overcome the plasma membrane barrier and enter cells directly, either alone or with associated cargoes. However, the cellular entry mechanisms of CPPs in cyanobacteria have not been studied.ResultsIn the present study, we determine CPP-mediated transduction efficiency and internalization mechanisms in cyanobacteria using a combination of biological and biophysical methods. We demonstrate that both Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942 strains of cyanobacteria possess red autofluorescence. Green fluorescent protein (GFP), either alone or noncovalently associated with a CPP comprised of nine arginine residues (R9/GFP complexes), entered cyanobacteria. The ATP-depleting inhibitor of classical endocytosis, N-ethylmaleimide (NEM), could block the spontaneous internalization of GFP, but not the transduction of R9/GFP complexes. Three specific inhibitors of macropinocytosis, cytochalasin D (CytD), 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), and wortmannin, reduced the efficiency of R9/GFP complex transduction, indicating that entry of R9/GFP complexes involves macropinocytosis. Both the 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT) and membrane leakage analyses confirmed that R9/GFP complexes were not toxic to the cyanobacteria, nor were the endocytic and macropinocytic inhibitors used in these studies.ConclusionsIn summary, we have demonstrated that cyanobacteria use classical endocytosis and macropinocytosis to internalize exogenous GFP and CPP/GFP proteins, respectively. Moreover, the CPP-mediated delivery system is not toxic to cyanobacteria, and can be used to investigate biological processes at the cellular level in this species. These results suggest that both endocytic and macropinocytic pathways can be used for efficient internalization of regular protein and CPP-mediated protein delivery in cyanobacteria, respectively.
Highlights
The plasma membrane plays an essential role in selective permeability, compartmentalization, osmotic balance, and cellular uptake
Mechanistic studies of protein transduction To demonstrate protein transduction in cyanobacteria, both 6803 and 7942 strains were treated with either green fluorescent protein (GFP) alone or R9/Green fluorescent protein (GFP) noncovalently complexed at a molecular ratio of 3:1
All of these inhibitors reduced the entry of GFP, indicating that endocytosis is the route for spontaneous GFP internalization (Additional file 1: Figure S1B)
Summary
The plasma membrane plays an essential role in selective permeability, compartmentalization, osmotic balance, and cellular uptake. Cyanobacteria, known as blue-green algae, are photosynthetic prokaryotes. They played a key role in the evolution of life on Earth, converting the early reducing atmosphere into an oxidizing one as they performed oxygenic photosynthesis [1]. Many peptide analogues of the basic amino acid-rich domain of the Tat protein were synthesized and evaluated for membrane transduction potential [11,12]. These positively charged, amphipathic peptides were termed cell-penetrating peptides (CPPs) or protein transduction domains (PTDs) [11,12,13]
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