Junguang Jiang

Regulation of EGFR nanocluster formation by ionic protein-lipid interaction

The abnormal activation of epidermal growth factor receptor (EGFR) is strongly associated with a variety of human cancers but the underlying molecular mechanism is not fully understood. By using direct stochastic optical reconstruction microscopy (dSTORM), we find that EGFR proteins form nanoclusters in the cell membrane of both normal lung epithelial cells and lung cancer cells, but the number and size of clusters significantly increase in lung cancer cells. The formation of EGFR clusters is mediated by the ionic interaction between the anionic lipid phosphatidylinositol-4,5-bisphosphate (PIP2) in the plasma membrane and the juxtamembrane (JM) region of EGFR. Disruption of EGFR clustering by PIP2 depletion or JM region mutation impairs EGFR activation and downstream signaling. Furthermore, JM region mutation in constitutively active EGFR mutant attenuates its capability of cell transformation. Collectively, our findings highlight the key roles of anionic phospholipids in EGFR signaling and function, and reveal a novel mechanism to explain the aberrant activation of EGFR in cancers.

Direct Evidence of Lipid Rafts by in situ Atomic Force Microscopy

Lipid rafts are membrane microdomains enriched with cholesterol, glycosphingolipids, and proteins. Although they are broadly presumed to play a pivotal role in various cellular functions, there are still fierce debates about the composition, functions, and even existence of lipid rafts. Here high-resolution and time-lapse in situ atomic force microscopy is used to directly confirm the existence of lipid rafts in native erythrocyte membranes. The results indicate some important aspects of lipid rafts: most of the lipid rafts are in the size range of 100-300 nm and have irregular shape; the detergent-resistant membranes consist of cholesterol microdomains and are not likely the same as the lipid rafts; cholesterol contributes significantly to the formation and stability of the protein domains; and Band III is an important protein of lipid rafts in the inner leaflet of erythrocyte membranes, indicating that lipid rafts are exactly the functional domains in plasma membrane. This work provides direct evidence of the presence, size, and main constitutive protein of lipid rafts at a resolution of a few nanometers, which will pave the way for studying their structure and functions in detail.

Electrostatic-assembly metallized nanoparticles network by DNA template.

Eighteen-nanometer gold and 3.5-nm silver colloidal particles closely packed by cetyltrimethylammonium bromide (CTAB) to form its positively charged shell. The DNA network was formed on a mica substrate firstly. Later, CTAB-capped gold or silver colloidal solutions were cast onto DNA network surface. It was found that the gold or silver nanoparticles metallized networks were formed owing to the electrostatic-driven template assembling of positive charge of CTAB-capped gold and silver particles on the negatively charged phosphate groups of DNA molecules by the characterizations of AFM, XPS and UV-vis. This method may provide a novel and simple way to studying nanoparticles assembly conjugating DNA molecules and offer some potential promising applications in nanocatalysis, nanoelectronics, and nanosensor on the basis of the fabricated metal nanoparticles network.

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.

Conformation change of horseradish peroxidase in lipid membrane

The electrochemical behavior of horseradish peroxidase (HRP) in the dimyristoyl phosphatidylcholine (DMPC) bilayer on the glassy carbon (GC) electrode was studied by cyclic voltammetry. The direct electron transfer of HRP was observed in the DMPC bilayer. Only a small cathodic peak was observed for HRP on the bare GC electrode. The electron transfer of HRP in the DMPC membrane is facilitated by DMPC membrane. UV-Vis and circular dichroism (CD) spectroscopy were used to study the interaction between HRP and DMPC membrane. On binding to the DMPC membrane the secondary structure of HRP remains unchanged while there is a substantial change in the conformation of the heme active site. Tapping mode atomic force microscopy (AFM) was first applied for the investigation on the structure of HRP adsorbed on supported phospholipid bilayer on the mica and on the bare mica. HRP molecules adsorb and aggregate on the mica without DMPC bilayer. The aggregation indicates an attractive interaction among the adsorbed molecules. The molecules are randomly distributed in the DMPC bilayer. The adsorption of HRP in the DMPC bilayer changes drastically the domains and defects in the DMPC bilayer due to a strong interaction between HRP and DMPC films.

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.

Fabrication of Metalloporphyrin-Polyoxometalyte Hybrid Film by Layer-by-Layer Method and Its Catalysis for Dioxygen Reduction

Through layer-by-layer method [tetrakis(N-methylpyridyl)porphyrinato] cobalt (CoTMPyP) and polyoxometalyte were alternately deposited on 4-aminobenzoic acid (4-ABA) modified glassy carbon electrode. The resulting organic-inorganic hybrid films were characterized by cyclic voltammetry (CV), UV/visible absorption spectroscopy, and atomic force microscopy (AFM). It was proved that the multilayer films are uniform and stable. CoTMPyP-containing multilayer films exhibit remarkable electrocatalytic activity for the reduction of O-2. Rotating disk electrode (RDE) voltammetry and rotating ring-disk electrode (RRDE) voltammetry confirm that P2W18/CoTMPyP multilayer films can catalyze the four-electron almost reduction of O-2 to water in pH > 4.0 buffer solution, while SiW12/CoTMPyP multilayer films catalyze about two-electron reduction of O-2 to H2O2 in pH 1 - 6 buffer solutions. The kinetic constants for O-2 reduction were comparatively investigated at P2W18/CoTMPyP and SiW12/CoTMPyP multilayer films electrodes.

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.

Structural insights into the negative regulation of BRI1 signaling by BRI1-interacting protein BKI1

Brassinosteroids (BRs) are essential steroid hormones that have crucial roles in plant growth and development. BRs are perceived by the cell-surface receptor-like kinase brassinosteroid insensitive 1 (BRI1). In the absence of BRs, the cytosolic kinase domain (KD) of BRI1 is inhibited by its auto-inhibitory carboxyl terminus, as well as by interacting with an inhibitor protein, BRI1 kinase inhibitor 1 (BKI1). How BR binding to the extracellular domain of BRI1 leads to activation of the KD and dissociation of BKI1 into the cytosol remains unclear. Here we report the crystal structure of BRI1 KD in complex with the interacting peptide derived from BKI1. We also provide biochemical evidence that BRI1-associated kinase 1 (BAK1) plays an essential role in initiating BR signaling. Steroid-dependent heterodimerization of BRI1 and BAK1 ectodomains brings their cytoplasmic KDs in the right orientation for competing with BKI1 and transphosphorylation.