Yuping Shan

Size-dependent endocytosis of single gold nanoparticles

Herein we investigate the size-dependent force of endocytosing single gold nanoparticles by HeLa cells. The results reveal that both the uptake and unbinding force values are dependent upon the size of gold nanoparticles.

Localization of Na+-K+ ATPases in Quasi-Native Cell Membranes

Na(+)-K(+) ATPases have been observed and located by in situ AFM and single molecule recognition technique, topography and recognition imaging (TREC) that is a unique technique to specifically identify single protein in complex during AFM imaging. Na(+)-K(+) ATPases were well distributed in the inner leaflet of cell membranes with about 10% aggregations in total recognized proteins. The height of Na(+)-K(+) ATPases measured by AFM is in the range of 12-14 nm, which is very consistent with the cryoelectron microscopy result. The unbinding force between Na(+)-K(+) ATPases in the membrane and anti-ATPases on the AFM tip is about 80 pN with the apparent loading rate at 40 nN/s. Our results show the first visualization of an essential membrane protein, Na(+)-K(+) ATPase, in quasi-native cell membranes and may be significant to reveal the interactions between Na(+)-K(+) ATPases and other membrane proteins at the molecular level.

Preparation of cell membranes for high resolution imaging by AFM

Studies of cell membrane structure by atomic force microscopy (AFM) have been limited because of the softness of cell membranes. Here, we utilize a new technique of sample preparation to lay red blood cell membranes on the top of a mica surface to obtain high resolution images by in-situ AFM on both sides of cell membranes. Our results indicate that the location of oligosaccharides and proteins in red blood cell membranes might be different from the current membrane model. The inner membrane leaflet is covered by dense proteins with fewer free lipids than expected. In contrast, the outer membrane leaflet is quite smooth; oligosaccharides and peptides supposed to protrude out of the outer membrane leaflet surface might be actually hidden in the middle of hydrophilic lipid heads; transmembrane proteins might form domains in the membranes revealed by PNGase F and trypsin digestion. Our result could be significant to interpret some functions about red blood cell membranes and guide to heal the blood diseases related to cell membranes.

Caveolae-mediated endocytosis of biocompatible gold nanoparticles in living Hela cells

Efficient intracellular delivery of gold nanoparticles (AuNPs) and unraveling the mechanism underlying the intracellular delivery are essential for advancing the applications of AuNPs toward in vivo imaging and therapeutic interventions. We employed fluorescence microscopy to investigate the internalization mechanism of small-size AuNPs by living Hela cells. Herein, we found that the caveolae-mediated endocytosis was the dominant pathway for the intracellular delivery of small-size AuNPs. The intracellular delivery was suppressed when we depleted the cholesterol with methyl-β-cyclodextrin (MβCD); in contrast, the sucrose that disrupts the formation of clathrin-mediated endocytosis did not block the endocytosis of AuNPs. Meanwhile, we examined the intracellular localization of AuNPs in endocytic vesicles by fluorescent colocalization. This work would provide a potential technique to study the intracellular delivery of small-size nanoparticles for biomedical applications.

Single-Particle Tracking of Hepatitis B Virus-like Vesicle Entry into Cells

HBsAg, the surface antigen of the hepatitis B virus (HBV), is used as a model to study the mechanisms and dynamics of a single-enveloped virus infecting living cells by imaging and tracking at the single-particle level. By monitoring the fluorescent indicator of HBsAg particles, it is found that HBsAg enters cells via a caveolin-mediated endocytic pathway. Tracking of individual HBsAg particles in living cells reveals the anomalously actin-dependent but not microtubule-dependent motility of the internalized HBsAg particle. The motility of HBsAg particles in living cells is also analyzed quantitatively. These results may settle the long-lasting debate of whether HBV directly breaks the plasma membrane barrier or relies on endocytosis to deliver its genome into the cell, and how the virus moves in the cell.

The study of single anticancer peptides interacting with HeLa cell membranes by single molecule force spectroscopy

To determine the effects of biophysical parameters (e.g. charge, hydrophobicity, helicity) of peptides on the mechanism of anticancer activity, we applied a single molecule technique-force spectroscopy based on atomic force microscope (AFM)-to study the interaction force at the single molecule level. The activity of the peptide and analogs against HeLa cells exhibited a strong correlation with the hydrophobicity of peptides. Our results indicated that the action mode between α-helical peptides and cancer cells was largely hydrophobicity-dependent.

Locating the Band III protein in quasi-native cell membranes

Band III is a key protein for the structure and function of red blood cell membranes. To date, the distribution and morphology of Band III in cell membranes is still unclear because of limited approaches. We applied Topography and RECognition imaging microscopy (TREC), which extends the application of atomic force microscopy (AFM) to recognize a single molecule in a biological complex, to visually locate a single Band III protein in quasi-native cell membranes by anti-Band III-functionalized AFM tips under physiological conditions. The Band III proteins are well distributed in the inner leaflet of cell membranes. The height of the whole Band III protein in cell membranes is in the range of 9–13 nm. The unbinding force between Band III in the membrane and anti-Band III on the AFM tip is about 70 pN with the loading rate at 40 nN/s. Our result is significant in revealing the location and morphology of Band III in the inner cell membrane at the molecular level.

Specificity and mechanism of action of alpha-helical membrane-active peptides interacting with model and biological membranes by single-molecule force spectroscopy

In this study, to systematically investigate the targeting specificity of membrane-active peptides on different types of cell membranes, we evaluated the effects of peptides on different large unilamellar vesicles mimicking prokaryotic, normal eukaryotic, and cancer cell membranes by single-molecule force spectroscopy and spectrum technology. We revealed that cationic membrane-active peptides can exclusively target negatively charged prokaryotic and cancer cell model membranes rather than normal eukaryotic cell model membranes. Using Acholeplasma laidlawii, 3T3-L1, and HeLa cells to represent prokaryotic cells, normal eukaryotic cells, and cancer cells in atomic force microscopy experiments, respectively, we further studied that the single-molecule targeting interaction between peptides and biological membranes. Antimicrobial and anticancer activities of peptides exhibited strong correlations with the interaction probability determined by single-molecule force spectroscopy, which illustrates strong correlations of peptide biological activities and peptide hydrophobicity and charge. Peptide specificity significantly depends on the lipid compositions of different cell membranes, which validates the de novo design of peptide therapeutics against bacteria and cancers.

Ultrafast Tracking of a Single Live Virion During the Invagination of a Cell Membrane

The first step in most viral infections is the penetration of the cell membrane via endocytosis. However, the underlying mechanism of this important process has not been quantitatively characterized; for example, the velocity and force of a single virion during invagination remain unknown. Here, the endocytosis of a single live virion (Singapore grouper iridovirus, SGIV) through the apical membranes of a host cell is monitored by developing and using a novel ultrafast (at the microsecond level) tracking technique: force tracing. For the first time, these results unambiguously reveal that the maximum velocity during the cell entry of a single SGIV by membrane invagination is approximately 200 nm s(-1), the endocytic force is approximately 60.8 ± 18.5 pN, and the binding energy density increases with the engulfment depth. This report utilizing high temporospatial resolution (subnanometer and microsecond levels) approaches provides new insight into the dynamic process of viral infection via endocytosis and the mechanism of membrane invagination at the single-particle level.

The force of transporting a single amino acid into the living cell measured using atomic force microscopy

We used single molecule force spectroscopy (SMFS) to investigate the interacting force between single cysteine and amino acid transporters in eukaryotic cell membranes. We measured the transporting force of cysteine and found that its conformation on the AFM tip is important for discriminating the substrate in the transporting pathway.