Saju Nettikadan

Molecular imaging of Escherichia coli F0F1-ATPase in reconstituted membranes using atomic force microscopy

The structure of Escherichia coli F0F1‐ATPase (ATP synthase), and its F0 sector reconstituted in lipid membranes was analyzed using atomic force microscopy (AFM) by tapping‐mode operation. The majority of F0F1‐ATPases were visualized as spheres with a calculated diameter of , and a height of from the membrane surface. F0 sectors were visualized as two different ring‐like structures (one with a central mass and the other with a central hollow of depth) with a calculated outer diameter of . The two different images possibly represent the opposite orientations of the complex in the membranes. The ring‐like projections of both images suggest inherently asymmetric assemblies of the subunits in the F0 sector. Considering the stoichiometry of F0 subunits, the area of the image observed is large enough to accommodate all three F0 subunits in an asymmetric manner.

Detection and Quantification of Protein Biomarkers from Fewer than 10 Cells

The use of antibody microarrays continues to grow rapidly due to the recent advances in proteomics and automation and the opportunity this combination creates for high throughput multiplexed analysis of protein biomarkers. However, a primary limitation of this technology is the lack of PCR-like amplification methods for proteins. Therefore, to realize the full potential of array-based protein biomarker screening it is necessary to construct assays that can detect and quantify protein biomarkers with very high sensitivity, in the femtomolar range, and from limited sample quantities. We describe here the construction of ultramicroarrays, combining the advantages of microarraying including multiplexing capabilities, higher throughput, and cost savings with the ability to screen very small sample volumes. Antibody ultramicroarrays for the detection of interleukin-6 and prostate-specific antigen (PSA), a widely used biomarker for prostate cancer screening, were constructed. These ultramicroarrays were found to have a high specificity and sensitivity with detection levels using purified proteins in the attomole range. Using these ultramicroarrays, we were able to detect PSA secreted from 100 LNCaP cells in 3 h and from just four LNCaP cells in 24 h. Cellular PSA could also be detected from the lysate of an average of just six cells. This strategy should enable proteomic analysis of materials that are available in very limited quantities such as those collected by laser capture microdissection, neonatal biopsy microspecimens, and forensic samples.

Label-Free Protein and Pathogen Detection Using the Atomic Force Microscope

The atomic force microscope (AFM) uses a sharp micron-scale tip to scan and amplify surface features, providing exceptionally detailed topographical information with magnification on the order of ×106. This instrument is used extensively for quality control in the computer and semiconductor industries and is becoming a progressively more important tool in the biological sciences. Advantages of the AFM for biological application include the ability to obtain information in a direct, label-free manner and the ability to image in solution, providing real-time data acquisition under physiologically relevant conditions. A novel application of the AFM currently under development combines its surface profiling capabilities with fixed immuno-capture using antibodies immobilized in a nanoarray format. This provides a distinctive platform for direct, label-free detection and characterization of viral particles and other pathogens.

ViriChip: a solid phase assay for detection and identification of viruses by atomic force microscopy

Bionanotechnology can be viewed as the integration of tools and concepts in nanotechnology with the attributes of biomolecules. We report here on an atomic force microscopy–immunosensor assay (AFMIA) that couples AFM with solid phase affinity capture of biological entities for the rapid detection and identification of group B coxsackievirus particles. Virus identification is based on type-specific immunocapture and the morphological properties of the captured viruses as obtained by the AFM. Representatives of the six group B coxsackieviruses have been specifically captured from 1 µl volumes of clarified cell lysates, body fluids and environmental samples. Concentration and kinetic profiles for capture indicate that detection is possible at 103 TCID50 µl−1 and the dynamic range of the assay spans three logs. The results demonstrate that the melding of a nanotechnological tool (AFM) with biotechnology (solid phase immunocapture of virus particles) can create a clinically relevant platform, useful for the detection and identification of enterovirus particles in a variety of samples.

Subcellular scaled multiplexed protein patterns for single cell cocultures

Tip-based direct protein printing is a relatively new technique that is useful for controlling the cellular microenvironment with subcellular resolution. Coculture studies have been useful for mimicking the in vivo environment and studying effects on stem or progenitor cell function. However, there are many experimental variables that cannot be properly controlled and may lead to confounding results. Here we demonstrate a technique that allows spatial control of multiple cell types at single cell levels on a substrate. Specifically, 3T3 fibroblasts and C2C12 myoblasts and their respective binding dynamics with fibronectin and laminin demonstrate the single cell coculture concept.

Molecular imaging of Na+,K÷-ATPase in purified kidney membranes

Ion channels and pumps in cell membranes consist of multiple transmembrane segments that are thought to be critical for transport of ions. Channel structures constituted by these transmembrane segments are characteristic of ion channels, whereas such structures have not been identified in ion pumps until now. By applying atomic force microscopy on Na+,K+‐ATPase molecules in canine kidney membranes under tapping mode, we identified a hollow in the protein with a characteristic internal diameter of 6–20A˚and an external diameter of 20–55A˚depending upon treatment conditions. This hollow may be interpreted as a channel‐like conformation of Na+,K+‐ATPase. In the regions where the proteins were absent, lipid head structures with 2A˚width and 6A˚length were imaged in an orthorhombic lattice.

CHARACTERIZATION OF TESTUDINE MELANOMACROPHAGE LINEAR, MEMBRANE EXTENSION PROCESSES—CABLEPODIA—BY PHASE AND ATOMIC FORCE MICROSCOPY

SummaryMelanomacrophages (MMs) are a component of an internal, pigmented cell system in liver and splenic tissues of some fishes, anurans, and reptiles. The cells have been found in centers or aggregates in sinusoids and are associated with cells capable of producing a peptide cytokine and immunoglobulins. A unique cell extension process has been observed in turtle MMs placed into cell culture, and this process has been studied by light and atomic force microscopy. These structures, referred to as cablepodia, are uniquely straight, narrow, and unbranching and appear to originate from growth cones opposite lamellipodia. Cablepodia were found to connect with other turtle MMs and fibroblasts forming cell networks. Dividing fibroblasts to which a cablepodium attached ceased cell division. The observations collectively suggest that a principal reason for aggregations of MMs in internal organs of lower vertebrates in their ability to form interconnected networks of cell processes for trapping and processing of particulate matter, cells and infectious organisms and, possibly, for the communication of cell signals and transfer of intracellular materials.

Negative transcriptional regulation of the chicken Na+/K+-ATPase α1-subunit gene

Abstract Although the Na + /K + -ATPase α1-subunit gene is ubiquitously expressed in vertebrates, its level of expression varies among tissue and cell types. In spite of similar mRNA distribution in tissues of mammals and birds, the 5′-flanking regions of α1-subunit genes exhibit remarkable diversity; i.e., the core promoter activity of the TATA-less chicken α1 gene strongly depends upon multiple Sp1-based regulation (six Sp1 sites), whereas the promoter activity of the TATA-like rat α1-subunit gene relies on the two Sp1 and additional positive regulatory factors. Further analysis of the regulatory regions of the Na + /K + -ATPase α1-subunit genes revealed that the vertebrate α1-subunit genes may share common inhibitory mechanisms for subtle transcriptional regulation; the core promoter activities can be either enhanced or repressed depending on the availability of inhibitory factors. Two potential candidates for such inhibitory elements in both avian and mammalian Na + /K + -ATPase α1-subunit genes are (1) a newly identified element, GCCCTC, and (2) a GCF-binding sequence, NN[G/c]CG[G/c][G/c][G/c]CN, or its reverse complement. Gel retardation assays using the inhibitory region of the chicken gene and crude nuclear extracts from tissue-cultured chicken and mouse cells showed the existence of a set of proteins that bind to this region. The amounts of individual regulatory proteins in different cell types seem to vary, resulting in differential formation of DNA/protein complexes in different cell types. Thus, the regulation of Na + /K + -ATPase α1-subunit gene expression under different cellular environment as well as in different cell types can be achieved by a shared mechanism; modulation of the ratio of the abundance of individual inhibitory factors.

Directed/localized growth of multiwalled carbon nanotubes catalyzed by cobalt nanoclusters

Tip based lithography was used to print arbitrary patterns of cobalt oxide nanoclusters from single features to large repeatable arrays over square millimeter areas. Cobalt ions were mixed with a P2VP-PEO block-copolymer and were delivered in sub-micron sized droplets to a surface. These droplets were heat treated at 360 °C, which removed the organic polymer and formed Co3O4 nanoclusters. The deposited material was used as a catalyst to grow multiwalled carbon nanotubes (MCNTs) at each printed location. AFM and SEM imaging were used to examine the morphology of the cobalt oxide nanoclusters and multiwalled carbon nanotubes. Powder X-ray diffraction was used to identify the catalytic material, while Raman spectroscopy was used to interrogate the resulting carbon structures. This directed multiwalled carbon nanotube growth method can be applied directly for sensor fabrication.