Topography and functional information of plasma membrane

Abstract

By using atomic force microscope (AFM), the topography and function of the plasmalemma surface of the isolated protoplasts from winter wheat mesophyll cells were observed, and compared with dead protoplasts induced by dehydrating stress. The observational results revealed that the plasma membrane of living protoplasts was in a state of polarization. Lipid layers of different cells and membrane areas exhibited distinct active states. The surfaces of plasma membranes were unequal, and were characterized of regionalisation. In addition, lattice structures were visualized in some regions of the membrane surface. These typical structures were assumed to be lipid molecular complexes, which were measured to be 15.8+/-0.09 nm in diameter and 1.9+/-0.3 nm in height. Both two-dimensional and three-dimensional imaging showed that the plasmalemma surfaces of winter wheat protoplasts were covered with numerous protruding particles. In order to determine the chemical nature of the protruding particles, living protoplasts were treated by proteolytic enzyme. Under the effect of enzyme, large particles became relatively looser, resulting that their width was increased and their height decreased. The results demonstrated that these particles were likely to be of protein nature. These protein particles at plasmalemma surface were different in size and unequal in distribution. The diameter of large protein particles ranged from 200 to 440 nm, with a central micropore, and the apparent height of them was found to vary from 12 to 40 nm. The diameter of mid-sized protein particles was between 40-60 nm, and a range of 1.8-5 nm was given for the apparent height of them. As for small protein particles, obtained values were 12-40 nm for their diameter and 0.7-2.2 nm for height. Some invaginated pits were also observed at the plasma membrane. They were formed by the endocytosis of protoplast. Distribution density of them at plasmalemma was about 16 pits per 15 microm(2). According to their size, we classified the invaginated pits into two types--larger pits measuring 139 nm in diameter and 7.2 nm in depth, and smaller pits measuring 96 nm in diameter and 2.3 nm in depth. On dehydration-induced dead protoplasts, the degree of polarization of plasma membranes decreased. Lipid molecular layers appeared relatively smooth, and the quantity of integral proteins reduced a lot. Invaginated pits were still detectable at the membrane surface, but due to dehydration-induced protoplast contraction, the orifice diameter of pits reduced, and their depth increased. Larger pits averagely measuring 47.4 nm in diameter and 31.9 nm in depth, and smaller pits measuring 26.5 nm in diameter and 43 nm in depth at average. The measured thickness of plasma membranes of mesophyll cells from winter wheat examined by AFM was 6.6-9.8 nm, thicker in regions covered with proteins.

Metallic Nanofilm Enhanced Fluorescence Cell Imaging: A Study of Distance-Dependent Intensity and Lifetime by Optical Sectioning Microscopy

“…For all dyes, the fluorescence image intensities on The cells were stained with different dyes at different subregions (DiI, PI for membrane and nucleus, and AO for DNA and RNA in both cytoplasm and nucleus, respectively). The thickness of the cell membrane is on the order of 10 nm, 42 and the thickness of the cell and cell cytoplasm was estimated to be 9 μm by measuring the section span from the basal membrane to the upper membrane of the DiI-stained cells, as shown in the Supporting Information (see Figure S3). DiI, AO-RNA, and PI, whose emission wavelengths are all in the red region, stain the cells at different positions.…”

Detecting CD20-Rituximab specific interactions on lymphoma cells using atomic force microscopy

“…Unlike other techniques, it can be applied to fluids, which makes observation of the dynamic physiology of living cells possible [7]. Additionally AFM has nanometer resolution [8], is easy to control, causes little damage to samples with small scan force [9], and the sample preparation is simple and fast [10]. Consequently, AFM has been widely applied in the investigation of cell physiology, and has provided new insights into biological activities at the cellular and molecular levels [11].…”