Membrane specific mapping and colocalization of malarial and host skeletal proteins in the Plasmodium falciparum infected erythrocyte by dual-color near-field scanning optical microscopy

Abstract

Accurate localization of proteins within the substructure of cells and cellular organelles enables better understanding of structure-function relationships, including elucidation of protein-protein interactions. We describe the use of a near-field scanning optical microscope (NSOM) to simultaneously map and detect colocalized proteins within a cell, with superresolution. The system we elected to study was that of human red blood cells invaded by the human malaria parasite Plasmodium falciparum. During intraerythrocytic growth, the parasite expresses proteins that are transported to the erythrocyte cell membrane. Association of parasite proteins with host skeletal proteins leads to modification of the erythrocyte membrane. We report on colocalization studies of parasite proteins with an erythrocyte skeletal protein. Host and parasite proteins were selectively labeled in indirect immunof luorescence antibody assays. Simultaneous dualcolor excitation and detection with NSOM provided f luorescence maps together with topography of the cell membrane with subwavelength (100 nm) resolution. Colocalization studies with laser scanning confocal microscopy provided lower resolution (310 nm) f luorescence maps of cross sections through the cell. Because the two excitation colors shared the exact same near-field aperture, the two f luorescence images were acquired in perfect, pixel-by-pixel registry, free from chromatic aberrations, which contaminate laser scanning confocal microscopy measurements. Colocalization studies of the protein pairs of mature parasite-infected erythrocyte surface antigen (MESA)(parasite)͞protein4.1(host) and P. falciparum histidine rich protein (PfHRP1)(parasite)͞ protein4.1(host) showed good real-space correlation for the MESA͞protein4.1 pair, but relatively poor correlation for the PfHRP1͞protein4.1 pair. These data imply that NSOM provides high resolution information on in situ interactions between proteins in biological membranes. This method of detecting colocalization of proteins in cellular structures may have general applicability in many areas of current biological research.One of the crucial aspects of current biological inquiry relates to the organization of cells and how interactions between proteins are involved in important cellular processes, such as signal transduction and receptor-ligand binding. Several experimental systems have been developed in recent years to try and identify such protein-protein interactions including the two hybrid and phage display systems (1, 2). All such attempts to identify interactions in disassembled cellular systems need to be confirmed in vivo, by approaches such as chemical cross-linking in cells or some form of colocalization study using microscopy. A common approach has been the use of confocal microscopy using fluorescent antibody probes. We report here the development of a technique based on dual-color immunofluorescence labeling together with near-field scanning optical microscopy (NSOM) (3, 4), which offers significant advances in the det...

Ultrahigh-resolution multicolor colocalization of single fluorescent probes

“…For the Axioskop (Fig. 1b), two-color APD images were constructed by overlaying the two independent channels (17,27). On the Axiovert (Fig.…”

“…For example, deconvolution algorithms do not have unique solutions, require subsequent signal processing, and have limited ability to compensate for aberrations. NSOM is limited to surfaces (such as cell membranes) (17). Moreover, NSOM is very demanding to operate and difficult to implement for hydrated samples.…”

“…Previously, we reported a related colocalization scheme of two different-color dyes using NSOM that forced two different-wavelength excitation lasers through the same near-field aperture (thus ensuring overlapping excitation volumes). This scheme was used to colocalize malaria parasite proteins in the membrane of infected red blood cells (17,26,27).…”

Quantum Optics: Colloidal Fluorescent Semiconductor Nanocrystals (Quantum Dots) in Single-Molecule Detection and Imaging

“…This can only be done by effectively reducing the volume of the PSF. Several methods have been proposed and demonstrated: (i) near-field scanning optical microscopy (NSOM; Betzig and Chichester (1993), Betzig and Trautman (1992); Enderle et al (1997) ) or interference methods such as (ii) standing-wave microscopy (Bailey et al 1993;Frohn et al 2000), (iii) I 5 M (Gustafsson 2005;Gustafsson et al 1999), (iv) 4Pi microscopy (Egner and Hell 2005;Hell 2003;Hell and Stelzer 1992), and (v) stimulated emission (STED) microscopy (Dyba and Hell 2002;Hofmann et al 2005;Klar et al 2000) although the applicability of some of these techniques remain to be demonstrated with QDs.…”

The interfacial bioscience grand challenge.

“…Integral membrane proteins pose a significant analytical challenge due to their amphiphillic nature and their tendency to aggregate and adhere to glass, plastic, and commonly used HPLC stationary phases. However, several groups have recently made progress in the preparation of integral membrane proteins, including rhodopsin, for mass spectrometry [26][27][28][29][30] . In our work, selected methods were optimized in order to maximize the recovery of cross-linked rhodopsin for subsequent LC-MS and tandem MS analysis.…”