Macropinocytosis: searching for an endocytic identity and role in the uptake of cell penetrating peptides

Abstract

Macropinocytosis defines a series of events initiated by extensive plasma membrane reorganization or ruffling to form an external macropinocytic structure that is then enclosed and internalized. The process is constitutive in some organisms and cell types but in others it is only pronounced after growth factor stimulation. Internalized macropinosomes share many features with phagosomes and both are distinguished from other forms of pinocytic vesicles by their large size, morphological heterogeneity and lack of coat structures. A paucity of information is available on other distinguishing features for macropinocytosis such as specific marker proteins and drugs that interfere with its mechanism over other endocytic processes. This has hampered efforts to characterize the dynamics of this pathway and to identify regulatory proteins that are expressed in order to allow it to proceed. Upon internalization, macropinosomes acquire regulatory proteins common to other endocytic pathways, suggesting that their identities as unique structures are short-lived. There is however less consensus regarding the overall fate of the macropinosome cargo or its limiting membrane and processes such as fusion, tubulation, recycling and regulated exocytosis have all been implicated in shaping the macropinosome and directing cargo traffic. Macropinocytosis has also been implicated in the internalization of cell penetrating peptides that are of significant interest to researchers aiming to utilize their translocation abilities to deliver therapeutic entities such as genes and proteins into cells. This review focuses on recent findings on the regulation of macropinocytosis, the intracellular fate of the macropinosome and discusses evidence for the role of this pathway as a mechanism of entry for cell penetrating peptides.

1

Endocytic pathways. A, caveolae delivering to caveosomes; B, clathrin independent endocytosis, C, clathrin coated uptake D, macropinocytosis showing macropinosome formation via fusion of ruffles with each other or the plasma membrane, E, phagocytosis. The contribution of a common early endosome in linking these pathways and partitioning membranes, proteins and cargo, over delivery to distinct classes of sorting endosomes as depicted by the dashed lines is unknown.

2

Localization of D-R8 in cells. HeLa (A) and A549 (B) cells were incubated for 1 hr at 37°C with 2 μM of the D-enantiomerof R8-Alexa488 peptide (RRRRRRRRGC-Alexa488) and immediately analysed by scanning confocal fluorescence microscopy combined with scanning Direct Interference Contrast. Shown are superimposed images from 25 to 30 sections through the Z-axis. The peptide-labelled vesicles in these cells are concentrated in a perinuclear region but it remains to be seen whether any are, or are derived from, macropinosomes. Previous studies in HeLa cells show a significant fraction of these to be late endosomes and lysosmes [110]. Scale bar 15 μM.

3

Proposed model for the cellular entry of cationic cell penetrating peptides HIV-TAT and R8. Uptake of the peptides occurs by at least two temperature and concentration dependent mechanisms [112]. At 4°C, entry can only occur via plasma membrane translocation. At 37°C and at high, threshold, extracellular concentration the peptide may enter the cell via a number of endocytic pathways and via direct plasma membrane translocation. A number of endocytic pathways including macropinocytosis caused by peptide induced plasma membrane ruffling is likely to be utilized. Escape from undefined endosomal compartments (?) could conceivably be the result of the accumulation of the peptide to a similar threshold concentration that promotes translocation across the plasma membrane.