Analytical Approaches for Studying Transporters, Channels and Porins

et al. 2015

Acta Pharmacol Sin

Knowledge of the nanoscale changes that take place in individual cells in response to a drug is useful for understanding the drug action. However, due to the lack of adequate techniques, such knowledge was scarce until the advent of atomic force microscopy (AFM), which is a multifunctional tool for investigating cellular behavior with nanometer resolution under near-physiological conditions. In the past decade, researchers have applied AFM to monitor the morphological and mechanical dynamics of individual cells following drug stimulation, yielding considerable novel insight into how the drug molecules affect an individual cell at the nanoscale. In this article we summarize the representative applications of AFM in characterization of drug actions on cell membrane, including topographic imaging, elasticity measurements, molecular interaction quantification, native membrane protein imaging and manipulation, etc. The challenges that are hampering the further development of AFM for studies of cellular activities are aslo discussed.

Section: Wwwchinapharcom LI M Et Almentioning

confidence: 99%

Section: High-resolution Imaging and Manipulation Of Individual Nativmentioning

confidence: 99%

Nanoscale monitoring of drug actions on cell membrane using atomic force microscopy

et al. 2015

Acta Pharmacol Sin

“…Thus membrane proteins can be reconstituted into lipid nanodiscs to examine their stability and folding; and the nanodiscs should permit high-throughput SMFS of membrane proteins. In 2009, Sapra et al [59] investigated the unfolding of the membrane protein OmpG that was reconstituted in lipids and revealed that the unfolding pathway was via amino acids 8,43,83,126,166,204, and 248. In 2012, Thoma et al [65] investigated the unfolding of membrane protein FhuA: after attaching the E. coli polar lipid bilayers containing reconstituted FhuA molecules onto freshly cleaved mica, AFM imaging was first performed to acquire the morphology of FhuA (Fig.…”

Section: Mechanically Unfolding Membrane Proteins With Smfsmentioning

confidence: 99%

Progress in measuring biophysical properties of membrane proteins with AFM single-molecule force spectroscopy

et al. 2014

Chin. Sci. Bull.

Membrane proteins are crucial in cell physiological activities and are the targets for most drugs. Thus, investigating the behaviors of membrane proteins not only provide deeper insights into cell function, but also help disease treatment and drug development. Atomic force microscopy is a unique tool for investigating the structure of membrane proteins. It can both image the morphology of single native membrane proteins with high resolution and, via single-molecule force spectroscopy (SMFS), directly measure their biophysical properties during molecular physiological activities such as ligand binding and protein unfolding. In the context of molecular biomechanics, SMFS has been successfully used to understand the structure and function of membrane proteins, complementing the static three-dimensional structures of proteins obtained by X-ray crystallography. Here, based on the authors' antigen-antibody binding force measurements in clinical tumor cells, the principle and method of SMFS is discussed, the progress in using SMFS to characterize membrane proteins is summarized, and challenges for SMFS are presented.

“…The majority of cells have many different membrane proteins, therefore protein expression techniques are needed to create a cell membrane that has only one type of protein. The main expression systems are bacterial, yeast, insect cells, and mammalian cells [6]. After the target proteins are expressed in the cell membrane, patches of the membranes are suspended and then attached onto a substrate for AFM imaging [19].…”

Section: Sample Preparation Techniquesmentioning

confidence: 99%

“…An exception is environmental scanning electron microscopy (ESEM), but, at present, the image resolution is low and is still unable to image living cells [4]. X-ray crystallography can only provide static three-dimensional structures of proteins and cannot observe proteins dynamics [5]; a truly functional protein is dynamic by nature [6]. AFM has nanometer spatial resolution, it is easy to control, it can serially observe in fluids the dynamics of living cells and native membrane proteins (including the micro-environments around the membrane protein), and sample preparation is relatively simple.…”

mentioning

confidence: 99%

Progress of AFM single-cell and single-molecule morphology imaging

et al. 2013

Chin. Sci. Bull.

Atomic force microscopy (AFM) can probe single living cells and single native membrane proteins in natural fluid environments with label-free high spatial resolution. It has thus become an important tool for cellular and molecular biology that significantly complements traditional biochemical and biophysical techniques such as optical and electron microscopy and X-ray crystallography. Imaging surface topography is the primary application of AFM in the life sciences. Since the early 1990s, researchers have used AFM to investigate morphological features of living cells and native membrane proteins with impressive results. Steady improvements in AFM techniques for imaging soft biological samples have greatly expanded its applications. Based on the authors' own research in AFM imaging of living cell morphologies, a review of sample preparation procedures for single-cell and single-molecule imaging experiments is presented, along with a summary of recent progress in AFM imaging of living cells and native membrane proteins. Finally, the challenges of AFM high-resolution imaging at the single-cell and single-molecule levels are discussed.