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Structural studies of filament-forming biomolecules have classically relied on optical imaging methods such as fluorescence and electron microscopy. While these methods have yielded novel insights into mechanisms of macromolecular assembly, they often require perturbative labeling or staining procedures that can affect the assembly process. In contrast, Atomic Force Microscopy (AFM) has emerged as a powerful imaging tool to directly study native nanostructures at high resolution without labeling. Unlike optical methods, AFM utilizes a nano-scale cantilever to generate a topography map of surface-immobilized molecules in air or in aqueous environments, allowing studies under more physiological or dynamic conditions. However, like all imaging methods, most biological samples require electrostatic or covalent interaction of the biomolecule with a surface for imaging, and care must be taken to avoid surface-specific effects on the sample. Here, we examined the structural organization of the bacterial polar scaffold protein PopZ by AFM, and analyzed the effect of surface-immobilization conditions on PopZ's nanostructural assembly characteristics. We measured the structures and densities of PopZ complexes on positively and negatively charged as well as hydrophobic surfaces, and compared structural organization of these assemblies in aqueous environments. Our results illustrate how choice of surface immobilization conditions can affect structural studies of polymeric assemblies, and demonstrate the tremendous advantages of AFM for directly imaging biomolecules in aqueous, physiological conditions. Finally, our results provide new insight into the structures of multimeric PopZ nano-assemblies that have been thus far unattainable using standard EM methods, providing direct evidence for PopZ self-assembly into organized three-dimensional polymeric networks.

Many protein-RNA complexes exist within cells and have various functions, including shuttling mRNA from the site of transcription to specific cellular regions. Often, these protein-RNA complexes consist of multiple mRNAs of which some are known and others remain unknown. The traditional method used to isolate protein-mRNA complexes is immunoprecipitation (IP). IP involves lysing a population of cells and isolating the protein-mRNA complexes using antibodies specific to the protein to "pulldown" the corresponding protein-mRNA complex of interest. We demonstrate the use of AFM as a complementary analysis tool for studying protein-RNA complexes in single living cells. While IP homogenizes the composition of protein-mRNA complexes from the cellular population under investigation and represents an "average", the AFM can interrogate not only a single cell but also subcellular regions of a single live cell, allowing stratification of protein-mRNA complexes from different regions of the cell. The result is the ability to spatially discriminate different populations of mRNAs that may be carried by the shuttling protein. To demonstrate extraction of a protein-mRNA complex, we selected the shuttling protein, Zipcode Binding Protein 1 (ZBP1) and its known associated mRNA, beta-actin, as a model system. Rat fibroblasts expressing a fusion protein, ZBP1-mCherry, were visualized using the fluorescent microscope of the AFM and then the cells of interest were punctured with an AFM tip conjugated with antibodies to ZBP1. Subsequently, the AFM tip was collected and analyzed for beta-actin mRNA using RT-PCR. BioAnalyzer results confirmed the extraction of beta-actin mRNA. This work shows that the AFM can be used as a tool to extract protein-RNA complexes from different regions of single living cells, potentially expanding the cell biologist's toolset.