To explore the real-time dynamic behavior of molecular transporters of the cell-penetrating-peptide (CPP) type on a
biological membrane, single fluorescently labeled oligoarginine conjugates were imaged interacting with the
plasma membrane of
Chinese hamster ovary (CHO) cells. The diffusional motion on the membrane, characterized by
single-molecule diffusion coefficient and residence time (tau R), defined as the time from the initial appearance of a
single-molecule spot on the membrane (from the
solution) to the time the single molecule disappears from the imaging focal plane, was observed for a fluorophore-labeled octaarginine (a model guanidinium-rich CPP) and compared with the corresponding values observed for a tetraarginine conjugate (negative control), a
lipid analogue, and a fluorescently labeled protein conjugate (transferrin-Alexa594) known to enter the cell through
endocytosis. Imaging of the oligoarginine conjugates was enabled by the use of a new high-contrast
fluorophore in the dicyanomethylenedihydrofuran family, which brightens upon interaction with the membrane at normal oxygen concentrations. Taken as a whole, the motions of the octaarginine conjugate single molecules are highly
heterogeneous and cannot be described as
Brownian motion with a single
diffusion coefficient. The observed behavior is also different from that of
lipids, known to penetrate
cellular membranes through
passive diffusion, conventionally involving lateral
diffusion followed by membrane
bilayer flip-flop. Furthermore, while the octaarginine conjugate behavior shares some common features with
transferrin uptake (endocytotic) processes, the two systems also exhibit dissimilar traits when diffusional motions and residence times of single constructs are compared. Additionally, pretreatment of cells with
cytochalasin D, a known
actin filament disruptor, produces no significant effect, which further rules out
unimodal endocytosis as the mechanism of uptake. Also, the involvement of
membrane potential in octaarginine-membrane interaction is supported by significant changes in the motion with high [K (+)] treatment. In sum, this first study of single transporter motion on the membrane of a
living cell indicates that the mode by which the octaarginine transporter penetrates the
cell membrane appears to either be a multimechanism uptake process or a mechanism different from
unimodal passive diffusion or
endocytosis.
