Nanogold Labeling Research Articles

Studies of functional properties of espin 1: Its interaction to actin filaments.

Actin is a multifunctional biomolecule that forms not only basic structural bodies such as filopodia and lamellipodia, but also large microvilli-like organelles like stereocilia. Actin consists of four sub-domains (S1, S2, S3, and S4), and the "target-binding groove" formed between S1 and S3 is the major binding site for various actin binding proteins. Actin filament dynamics are regulated by numerous actin binding proteins with different mechanisms of actin binding, assembly, and disassembly such as actin severing, branching, and bundling. Ectoplasmic specialization protein 1 (espin 1) is an actin binding and bundling protein that is specifically implicated in the elongation and stabilization of stereocilia as a binding partner with myosin III. However, little is known about the molecular structure, actin bundling, and stabilizing mechanism of espin 1; hence, we investigated the interaction between actin and espin 1 through structural data. In this study, we first purified human espin 1 in an E. coli system following a new detergent-free approach and then demonstrated the 2D structure of full-length espin 1 using transmission electron microscopy along with Nickel nitrilotriacetic acid nanogold labeling and 2D averaging using SPIDER. Furthermore, we also determined the espin 1 binding domain of actin using a co-sedimentation assay along with gelsolin and myosin S1. These findings are not only beneficial for understanding the actin binding and bundling mechanism of espin 1, but also shed light on its elongation, stabilization, and tip-localization mechanisms with myosin III. This study thus provides a basis for understanding the molecular structure of espin 1 and can contribute to various hearing-related diseases, such as hearing loss and vestibular dysfunction.

Abstract 1740: Determining the intracellular fate of ligand-conjugated therapeutics using nanogold labeling

Abstract Recent developments in ligand-conjugated RNAi therapeutics have led to improved stability of RNAi molecules, reducing effective therapeutic dose, and avoiding delivery vehicle-associated toxicity. However, as our group has shown in previous studies with FolamiR (Folate-conjugated miR-34a), endosomal entrapment of the therapeutic molecule is a rate-limiting factor in RNAi activity. To improve endosomal release of ligand-conjugates, several approaches are being explored such as incorporation of endosomal escape agents (nigericin), chemical modification to enhance stability of miRNA/siRNA within the acidic compartments, and modification of ligand-receptor interaction to enable adequate release from the receptor upon internalization. Current fluorescent microscopy techniques are only able to approximately estimate the cellular fate of FolamiR without providing enough resolution to determine the endosomal stage, receptor disengagement and other dynamics involved in the endosomal entrapment/release of FolamiR. To overcome this, we developed Folate-conjugated nanogold construct (FolaGold) that mimics FolamiR molecule. The nanogold in FolaGold is used as a proxy for miR-34a and electron microscopy can be performed to pinpoint the cellular fate of nanogold including receptor disengagement process, the endosomal stage as well as cytosolic enrichment at the nanometer resolution. In this study, FolaGold is validated to bind to folate receptor in a competition assay and is confirmed to be present in specific endosomal compartments in MB231 cells, unlike unconjugated nanogold. We further plan to use FolaGold to determine changes in intracellular fate of nanogold upon incorporation of endosomal escape agents (nigericin) and modification of ligand-receptor interaction by using a lower affinity binding ligand that facilitates ligand-receptor disengagement following internalization. While we are using nanogold labeling to determine the intracellular fate of ligand-conjugated RNAi therapeutics, the labeling can also help improve our understanding of endosomal release dynamics and activity of other ligand-conjugate based therapeutics. Citation Format: Ikjot S. Sohal, Ahmed M. Abdelaal, Andrea L. Kasinski. Determining the intracellular fate of ligand-conjugated therapeutics using nanogold labeling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1740.

Cell image 3D reconstruction based on nanogold labeling

Traditional cell image three-dimensional (3D) reconstruction methods have low accuracy, so a cell image 3D reconstruction method based on nanogold labeling was designed. Firstly, a cell image was collected, and the cell image was preprocessed and segmented. Then, the cell contour was extracted. Finally, the spatial distribution of nanogold cells was marked to complete the 3D reconstruction of the cell image. The experimental results show that the designed cell image 3D reconstruction method based on nanogold labeling is more accurate than the traditional method and can meet the needs of cell image 3D reconstruction.

Site-specific covalent labeling of large RNAs with nanoparticles empowered by expanded genetic alphabet transcription

Conjugation of RNAs with nanoparticles (NPs) is of significant importance because of numerous applications in biology and medicine, which, however, remains challenging especially for large ones. So far, the majority of RNA labeling relies on solid-phase chemical synthesis, which is generally limited to RNAs smaller than 100 nucleotides (nts). We, here, present an efficient and generally applicable labeling strategy for site-specific covalent conjugation of large RNAs with a gold nanoparticle (Nanogold) empowered by transcription of an expanded genetic alphabet containing the A-T/U and G-C natural base pairs (bps) and the TPT3-NaM unnatural base pair (UBP). We synthesize an amine-derivatized TPT3 (TPT3A), which is site specifically incorporated into a 97-nt 3'SL RNA and a 719-nt minigenomic RNA (DENV-mini) from Dengue virus serotype 2 (DENV2) by in vitro T7 transcription. The TPT3A-modified RNAs are covalently conjugated with mono-Sulfo-N-hydroxysuccinimidyl (NHS)-Nanogold NPs via an amine and NHS ester reaction and further purified under nondenaturing conditions. TPT3 modification and Nanogold labeling cause minimal structural perturbations to the RNAs by circular dichroism, small angle X-ray scattering (SAXS), and binding activity assay. We demonstrate the application of the Nanogold-RNA conjugates in large RNA structural biology by an emerging molecular ruler, X-ray scattering interferometry (XSI). The internanoparticle distance distributions in the 3'SL and DENV-mini RNAs derived from XSI measurements support the hypothetical model of flavivirus genome circularization, thus, validate the applicability of this labeling strategy. The presented strategy overcomes the size constraints in conventional RNA labeling strategies and is expected to have wide applications in large RNA structural biology and RNA nanotechnology.

Chimeric VP1-PCV2bCap protein as antigen for construction of recombinant vaccine against porcine circovirus type 2

Abstract Porcine circovirus type 2 (PCV2) is the main causative agents of post-weaning multi-systemic wasting syndrome leading to significant economic losses in swine population. Although vaccination is the most effective strategy to decrease morbidity and mortality rate, the virus persist in immunized populations and causes subclinical infections with symptoms similar to porcine circovirus diseases. To develop new effective vaccine against PCV2 infection, we prepared chimeric complex VP1-PCV2bCap containing PCV2bCap protein fused with mouse polyomavirus (MPyV). Complex was expressed in baculovirus expression system and purified by nickel agarose. From eluate containing large aggregates of purified proteins, fraction containing pantameres of VP1-PCV2bCap protein was separated by Asymmetrical flow field-flow fractionation and fractions were analyzed by electron microscopy with nano-gold labeling of PCV2Cap portion of the fused protein. 6 weeks old BALB/c mice were subcutaneously vaccinated by 50ug of purified chimeric protein alone or in combination with incomplete Freund adjuvants (50%) or Emulsigen (10%). Immunity response was monitored by indirect ELISA. After the first immunization strong immunity response that gradually increased after the second and third immunization was detected in all mice immunized by chimeric protein. The best immunity response was detected in mice immunized by vaccine with Emulsigen (10%) as adjuvant. Purified chimeric protein VP1-PCV2bCap has a high potential as a DIVA (differentiating infected form vaccinated animals) vaccine against PCV2b infections in pigs. The biggest benefit of this type of vaccine is possibility to distinguish between naturally infected and vaccinated animals

Conformational diversity of dynactin sidearm and domain organization of its subunit p150.

Dynactin is a principal regulator of the minus-end directed microtubule motor dynein. The sidearm of dynactin is essential for binding to microtubules and regulation of dynein activity. Although our understanding of the structure of the dynactin backbone (Arp1 rod) has greatly improved recently, structural details of the sidearm subcomplex remain elusive. Here, we report the flexible nature and diverse conformations of dynactin sidearm observed by electron microscopy. Using nanogold labeling and deletion mutant analysis, we determined the domain organization of the largest subunit p150 and discovered that its coiled-coil (CC1), dynein-binding domain, adopted either a folded or an extended form. Furthermore, the entire sidearm exhibited several characteristic forms, and the equilibrium among them depended on salt concentrations. These conformational diversities of the dynactin complex provide clues to understanding how it binds to microtubules and regulates dynein.

Structural organization of the C1a-e-c supercomplex within the ciliary central apparatus.

Nearly all motile cilia contain a central apparatus (CA) composed of two connected singlet microtubules with attached projections that play crucial roles in regulating ciliary motility. Defects in CA assembly usually result in motility-impaired or paralyzed cilia, which in humans causes disease. Despite their importance, the protein composition and functions of the CA projections are largely unknown. Here, we integrated biochemical and genetic approaches with cryo-electron tomography to compare the CA of wild-type Chlamydomonas with CA mutants. We identified a large (>2 MD) complex, the C1a-e-c supercomplex, that requires the PF16 protein for assembly and contains the CA components FAP76, FAP81, FAP92, and FAP216. We localized these subunits within the supercomplex using nanogold labeling and show that loss of any one of them results in impaired ciliary motility. These data provide insight into the subunit organization and 3D structure of the CA, which is a prerequisite for understanding the molecular mechanisms by which the CA regulates ciliary beating.

A single class of ARF GTPase activated by several pathway-specific ARF-GEFs regulates essential membrane traffic in Arabidopsis.

In eukaryotes, GTP-bound ARF GTPases promote intracellular membrane traffic by mediating the recruitment of coat proteins, which in turn sort cargo proteins into the forming membrane vesicles. Mammals employ several classes of ARF GTPases which are activated by different ARF guanine-nucleotide exchange factors (ARF-GEFs). In contrast, flowering plants only encode evolutionarily conserved ARF1 GTPases (class I) but not the other classes II and III known from mammals, as suggested by phylogenetic analysis of ARF family members across the five major clades of eukaryotes. Instead, flowering plants express plant-specific putative ARF GTPases such as ARFA and ARFB, in addition to evolutionarily conserved ARF-LIKE (ARL) proteins. Here we show that all eight ARF-GEFs of Arabidopsis interact with the same ARF1 GTPase, whereas only a subset of post-Golgi ARF-GEFs also interacts with ARFA, as assayed by immunoprecipitation. Both ARF1 and ARFA were detected at the Golgi stacks and the trans-Golgi network (TGN) by both live-imaging with the confocal microscope and nano-gold labeling followed by EM analysis. ARFB representing another plant-specific putative ARF GTPase was detected at both the plasma membrane and the TGN. The activation-impaired form (T31N) of ARF1, but neither ARFA nor ARFB, interfered with development, although ARFA-T31N interfered, like ARF1-T31N, with the GDP-GTP exchange. Mutant plants lacking both ARFA and ARFB transcripts were viable, suggesting that ARF1 is sufficient for all essential trafficking pathways under laboratory conditions. Detailed imaging of molecular markers revealed that ARF1 mediated all known trafficking pathways whereas ARFA was not essential to any major pathway. In contrast, the hydrolysis-impaired form (Q71L) of both ARF1 and ARFA, but not ARFB, had deleterious effects on development and various trafficking pathways. However, the deleterious effects of ARFA-Q71L were abolished by ARFA-T31N inhibiting cognate ARF-GEFs, both in cis (ARFA-T31N,Q71L) and in trans (ARFA-T31N + ARFA-Q71L), suggesting indirect effects of ARFA-Q71L on ARF1-mediated trafficking. The deleterious effects of ARFA-Q71L were also suppressed by strong over-expression of ARF1, which was consistent with a subset of BIG1-4 ARF-GEFs interacting with both ARF1 and ARFA. Indeed, the SEC7 domain of BIG5 activated both ARF1 and ARFA whereas the SEC7 domain of BIG3 only activated ARF1. Furthermore, ARFA-T31N impaired root growth if ARF1-specific BIG3 was knocked out and only ARF1- and ARFA-activating BIG4 was functional. Activated ARF1 recruits different coat proteins to different endomembrane compartments, depending on its activation by different ARF-GEFs. Unlike ARF GTPases, ARF-GEFs not only localize at distinct compartments but also regulate specific trafficking pathways, suggesting that ARF-GEFs might play specific roles in traffic regulation beyond the activation of ARF1 by GDP-GTP exchange.

Quantification of Aquaporin-Z reconstituted into vesicles for biomimetic membrane fabrication

Aquaporin incorporated biomimetic membranes are anticipated to offer unprecedented desalination capabilities. However, the lack of accurate methods to quantify the reconstituted aquaporin presents a huge hurdle in investigating aquaporin performance and optimizing membrane fabrication. Herein, we present three quantification methods to determine the Aquaporin-Z reconstituted into E. coli lipid vesicles: 1) nanogold labeling with transmission electron microscopy (TEM) visualization, 2) nickel labeling with inductively coupled plasma-mass spectrometry (ICP-MS) and 3) gel electrophoresis. The TEM method serves as a quick way to determine if aquaporin has been reconstituted, but is not quantitative. The numerical results from quantitative methods, ICP-MS and gel electrophoresis, correlate closely, showing that 60 ± 20% vs 66 ± 4% of Aquaporin-Z added is successfully reconstituted into vesicles respectively. These methods allow more accurate determination of Aquaporin-Z reconstituted and loss during reconstitution, with relatively commonly available equipment and without complex sample handling, or lengthy data analysis. These would allow them to be widely applicable to scientific studies of protein function in the biomimetic environment and engineering studies on biomimetic membrane fabrication.

Correlative Ultrastructural Analysis of Functionally Modulated Synapses Using Automatic Tape-Collecting Ultramicrotome - SEM Array Tomography

To understand synaptic plasticity, correlative microscopic approaches using functional imaging by light microscopy combined with electron microscopy (EM) are necessary. Ultrastructure of synapses can be visualized only by EM, but re-finding the same synapses that were manipulated and imaged by light microscope in the EM is a very laborious and time-consuming task when performing the conventional serial ultrathin-sectioning and observation method with transmission EM. Recently, new imaging applications to obtain volume data sets from brain tissues were introduced using an automatic tapecollecting microtome (ATUM) [1] or a focused ion beam [2] with scanning electron microscopy (SEM). We used an ATUMtome (RMC/Boeckeler) and automatic array tomography software, Atlas AT5 (Zeiss) and established a workflow to investigate ultrastructural changes of synapses. We first performed glutamate uncaging by 2-photon laser microscope to induce/modify spines, and obtained the whole cell image with a confocal microscope. Then the tissues are prepared with conventional pre-embedding immuno-EM using nanogold labeling for EM visualization as explained below. 2-photon glutamate uncaging and confocal laser scanning microscopy (CLSM) imaging: We uncaged MNI-glutamate with 4 mW (720 nm, 2 photon), 2 Hz X 40 stimulation on smooth dendrites of pyramidal neurons expressing EGFP in mouse hippocampal organotypic slice cultures. After the new spine formation visually confirmed, tissue slices were fixed in a mixture of 4% paraformaldehyde and 0.5-1% glutaraldehyde. CLSM Z-stack images were captured with a lower magnification to overview the whole tissue slice containing GFP fluorescent cells. Once the target neuron was identified, it was imaged to visualize its dendritic arbor and to define the position of the newly developed spines. Immuno-staining and resin embedding: Slices were cryo-protected with sucrose, permeabilized by liquid nitrogen, blocked and immunostained with anti-GFP antibody followed by nanogold conjugated secondary antibody. Slices were then silver enhanced, post-fixed with OsO4, en-block stained with uranyl acetate, dehydrated with a series of ethanol and acetone, and embedded in Durcupan resin. ATUM and SEM imaging with Atlas AT5/ Merlin VP Compact (Zeiss): The area containing the target cell was trimmed based on the CLSM image to a size of 1 x 1.5 mm. The sample was sectioned at 60-70 nm thickness with an ATUMtome and collected on Kapton tape automatically, then the kapton tape was placed on silicon wafers. The overview image of the Si wafer was captured using a digital camera, the image was loaded into the Atlas AT5 and aligned to the physical location of the wafer in the SEM using mesh grids as fiducials on the wafer. Low-resolution SE2 images for all sections were first captured with a 2 µm/pixel xy-resolution protocol (4 mm 2 imaging area) and overlaid on the overview image. An approximate area of the target cell was demarcated on the serial sections, and montage images were captured with a 30 nm pixel resolution protocol using BSE. After finding the target cell, a rough 3D reconstruction was performed to see the orientation of the target dendrite and spine. The high-resolution images of the target spine were captured with a 4-7 nm pixel resolution protocol using an InlensDuo detector in the BSE mode.

The Habc Domain of the SNARE Vam3 Interacts with the HOPS Tethering Complex to Facilitate Vacuole Fusion

Membrane fusion at vacuoles requires a consecutive action of the HOPS tethering complex, which is recruited by the Rab GTPase Ypt7, and vacuolar SNAREs to drive membrane fusion. It is assumed that the Sec1/Munc18-like Vps33 within the HOPS complex is largely responsible for SNARE chaperoning. Here, we present direct evidence for HOPS binding to SNAREs and the Habc domain of the Vam3 SNARE protein, which may explain its function during fusion. We show that HOPS interacts strongly with the Vam3 Habc domain, assembled Q-SNAREs, and the R-SNARE Ykt6, but not the Q-SNARE Vti1 or the Vam3 SNARE domain. Electron microscopy combined with Nanogold labeling reveals that the binding sites for vacuolar SNAREs and the Habc domain are located in the large head of the HOPS complex, where Vps16 and Vps33 have been identified before. Competition experiments suggest that HOPS bound to the Habc domain can still interact with assembled Q-SNAREs, whereas Q-SNARE binding prevents recognition of the Habc domain. In agreement, membranes carrying Vam3ΔHabc fuse poorly unless an excess of HOPS is provided. These data suggest that the Habc domain of Vam3 facilitates the assembly of the HOPS/SNARE machinery at fusion sites and thus supports efficient membrane fusion.

Nanogold Immunodetection Detection Systems for the Identification of Autoantigens by Western Blotting.

Nanogold-conjugated immunodetection systems are now widely and commercially available for use in a number of research applications including electron microscopy, light microscopy, and western blotting. Nanogold clusters are small, uniform in size, and stable, unlike gold colloids historically used in protein detection. Covalent linkage of nanogold particles to secondary antibodies prevents dissociation of the gold particles during the staining process, making protein detection reliable, antigen specific, and highly sensitive. Nanogold labeling is extremely versatile and can be used in conjunction with other staining methodologies including Alexa Fluor immunofluorescence detection to perform coupled staining procedures. Silver enhancement increases the limits of sensitivity for nanogold staining, thus improving detection signals for antigens with reduced expression levels. Herein, we describe the use of nanogold-silver detection as an immunodetection system for standard western blotting of autoantigens.

Redefining the modular organization of the core Mediator complex.

The Mediator complex plays an essential role in the regulation of eukaryotic transcription. The Saccharomyces cerevisiae core Mediator comprises 21 subunits, which are organized into Head, Middle and Tail modules. Previously, the Head module was assigned to a distinct dense domain at the base, and the Middle and Tail modules were identified to form a tight structure above the Head module, which apparently contradicted findings from many biochemical and functional studies. Here, we compared the structures of the core Mediator and its subcomplexes, especially the first 3D structure of the Head + Middle modules, which permitted an unambiguous assignment of the three modules. Furthermore, nanogold labeling pinpointing four Mediator subunits from different modules conclusively validated the modular assignment, in which the Head and Middle modules fold back on one another and form the upper portion of the core Mediator, while the Tail module forms a distinct dense domain at the base. The new modular model of the core Mediator has reconciled the previous inconsistencies between the structurally and functionally defined Mediator modules. Collectively, these analyses completely redefine the modular organization of the core Mediator, which allow us to integrate the structural and functional information into a coherent mechanism for the Mediator's modularity and regulation in transcription initiation.

Coordinated Rearrangements between Cytoplasmic and Periplasmic Domains of the Membrane Protein Complex ExbB-ExbD of Escherichia coli

Gram-negative bacteria rely on the ExbB-ExbD-TonB system for the import of essential nutrients. Despite decades of research, the stoichiometry, subunit organization, and mechanism of action of the membrane proteins of the Ton system remain unclear. We copurified ExbB with ExbD as an ∼240kDa protein-detergent complex, measured by light scattering and by native gels. Quantitative Coomassie staining revealed a stoichiometry of ExbB4-ExbD2. Negative stain electron microscopy and 2D analysis showed particles of ∼10nm diameter in multiple structural states. Nanogold labeling identified the position of the ExbD periplasmic domain. Random conical tilt was used to reconstruct the particles in three structural states followed by sorting of the single particles and refinement of each state. The different states are interpreted by coordinated structural rearrangements between the cytoplasmic domain and the periplasmic domain, concordant with invivo predictions.

Positively charged nanogold label allows the observation of fine cell filopodia and flagella in solution by atmospheric scanning electron microscopy

Optical microscopy is generally the first choice to observe microbes and cells. However, its resolution is not always sufficient to reveal specific target structures, such as flagella and pili, which are only nanometers wide. ASEM is an attractive higher resolution alternative, as the sample is observed in aqueous solution at atmospheric pressure. Sample pretreatment for ASEM only comprises simple tasks including fixation, gold labeling, and reagent exchange, taking less than 1 h in total. The lengthy sample pretreatments often required for more classical electron microscopies, such as embedding and dehydration, are unnecessary, and native morphology is preserved. In this study, positively charged nanogold particles were used to label the surfaces of bacteria and cultured animal cells, exploiting their net negative surface charge. After gold enhancement to increase the size of the nanogold particles, ASEM imaging of the bacteria in aqueous solution revealed pili and delicate spiral flagella. This natural shape contrasts starkly with images of dried flagella recorded by standard SEM. Positively charged nanogold labeled the plasma membrane of cultured COS7 cells, and after enhancement allowed filopodia as thin as 100 nm in diameter to be clearly visualized. Based on these studies, ASEM combined with positively charged nanogold labeling promises to become an important tool for the study of cell morphology and dynamics in the near future.

A visual detection method for Salmonella Typhimurium based on aptamer recognition and nanogold labeling

A rapid, specific and visible detection method for Salmonella Typhimurium was developed based on the recognition of aptamers coupled with nanogold labeling and silver signal amplification. Briefly, biotinylated aptamer 1, which was specific to Salmonella Typhimurium, was immobilized onto the surface of microtiter plate-wells modified with streptavidin through the binding of biotin and streptavidin. Then, the target bacteria, Salmonella Typhimurium, and the aptamer 2-gold nanoparticle (aptamer 2-AuNPs) conjugates were incubated in the wells to form sandwich-type aptamer/bacteria/aptamer-AuNPs complexes. Under optimal conditions, the correlation between the concentration of Salmonella Typhimurium and the intensity of the signals was observed to be linear within the range of 10–106 cfu mL−1 (R2 = 0.9913), and the detection limit of the proposed method was observed to be 7 cfu mL−1 This developed method offers the potential for rapid, sensitive and visible detection of Salmonella Typhimurium in samples.

Study and evaluation of Wondfo rapid diagnostic kit based on nano-gold immunochromatography assay for diagnosis of Plasmodium falciparum

Malaria has been recognized as a human disease for thousands of years and remains one of the most common diseases affecting humans worldwide. Therefore, a method for rapidly detecting Plasmodium falciparum is necessary and useful. We have developed Wondfo rapid diagnostic kit based on nano-gold immunochromatography assay for the detection of P. falciparum in patient specimen. In the present study, we demonstrated the sensitivity and specificity of the rapid diagnostic kit in which nano-gold labeling techniques and the monoclonal antibodies against histidine-rich protein-2 of P. falciparum were used to establish two-antibody sandwich immunochromatographic assay for detecting P. falciparum. By using microscopic examination of blood smears as control, the sensitivity, specificity, and feasibility of Wondfo rapid diagnostic kit was determined in the prompt and accurate diagnosis of malaria. In this study, 1,558 blood samples were collected from outpatient clinics in China and detected by both Wondfo kit and microscopic examination. The Wondfo kit did not show cross-reaction with microfilaria, Toxoplasma gondii, and other parasites in the blood. The patient samples positive for rheumatoid factor, HIV, tuberculosis, and syphilis did not show false positivity when testing with Wondfo kit. The detection sensitivity and specificity of Wondfo rapid diagnostic kit were 95.49% and 99.53%, respectively. These results indicate that our rapid diagnostic assay may be useful for detecting P. falciparum in patient specimen.

Analysis of the Intact Surface Layer of Caulobacter crescentus by Cryo-Electron Tomography

The surface layers (S layers) of those bacteria and archaea that elaborate these crystalline structures have been studied for 40 years. However, most structural analysis has been based on electron microscopy of negatively stained S-layer fragments separated from cells, which can introduce staining artifacts and allow rearrangement of structures prone to self-assemble. We present a quantitative analysis of the structure and organization of the S layer on intact growing cells of the Gram-negative bacterium Caulobacter crescentus using cryo-electron tomography (CET) and statistical image processing. Instead of the expected long-range order, we observed different regions with hexagonally organized subunits exhibiting short-range order and a broad distribution of periodicities. Also, areas of stacked double layers were found, and these increased in extent when the S-layer protein (RsaA) expression level was elevated by addition of multiple rsaA copies. Finally, we combined high-resolution amino acid residue-specific Nanogold labeling and subtomogram averaging of CET volumes to improve our understanding of the correlation between the linear protein sequence and the structure at the 2-nm level of resolution that is presently available. The results support the view that the U-shaped RsaA monomer predicted from negative-stain tomography proceeds from the N terminus at one vertex, corresponding to the axis of 3-fold symmetry, to the C terminus at the opposite vertex, which forms the prominent 6-fold symmetry axis. Such information will help future efforts to analyze subunit interactions and guide selection of internal sites for display of heterologous protein segments.

P22 Coat Protein Structures Reveal a Novel Mechanism for Capsid Maturation: Stability without Auxiliary Proteins or Chemical Crosslinks

Viral capsid assembly and stability in tailed, dsDNA phage and Herpesviridae are achieved by various means including chemical crosslinks (unique to HK97), or auxiliary proteins (lambda, T4, phi29, and herpesviruses). All these viruses have coat proteins (CP) with a conserved, HK97-like core structure. We used a combination of trypsin digestion, gold labeling, cryo-electron microscopy, 3D image reconstruction, and comparative modeling to derive two independent, pseudoatomic models of bacteriophage P22 CP: before and after maturation. P22 capsid stabilization results from intersubunit interactions among N-terminal helices and an extensive "P loop," which obviate the need for crosslinks or auxiliary proteins. P22 CP also has a telokin-like Ig domain that likely stabilizes the monomer fold so that assembly may proceed via individual subunit addition rather than via preformed capsomers as occurs in HK97. Hence, the P22 CP structure may be a paradigm for understanding how monomers assemble in viruses like phi29 and HSV-1.

FIZ1 is part of the regulatory protein complex on active photoreceptor-specific gene promoters in vivo

BackgroundFIZ1 (Flt-3 Interacting Zinc-finger) is a broadly expressed protein of unknown function. We reported previously that in the mammalian retina, FIZ1 interacts with NRL (Neural-Retina Leucine-zipper), an essential transcriptional activator of rod photoreceptor-specific genes. The concentration of FIZ1 in the retina increases during photoreceptor terminal maturation, when two key transcription factors NRL and CRX (Cone-Rod Homeobox) become detectable on the promoters of photoreceptor-specific genes (i.e. Rhodopsin, Pde6b). To determine if FIZ1 is involved in regulating CRX-mediated transcriptional activation, we examined FIZ1 subcellular location in mouse neural retina, its ability to interact with CRX, and its association with CRX/NRL target genes.ResultsFIZ1 is present in the nucleus of adult photoreceptors as well as other retinal neurons as shown by transmission electron microscopy with nano-gold labeling. FIZ1 and CRX were co-precipitated from retinal nuclear extracts with antibodies to either protein. Chromatin immunoprecipitation (ChIP) assays revealed that FIZ1 is part of the protein complex on several rod and cone gene promoters, within photoreceptor cells of the mouse retina. FIZ1 complexes with CRX or NRL on known NRL- and CRX-responsive elements, as shown by electrophoretic mobility shift assays with FIZ1 antibody. FIZ1 can directly bind to CRX, as demonstrated using yeast two-hybrid and GST pull-down assays. Co-transfection assays demonstrated that FIZ1 increases CRX-mediated activation of Opsin test promoters. Quantitative ChIP analysis revealed an increased association of FIZ1 with the Rhodopsin promoter in adult (P-25) neural retina versus immature (P-3) neural retina. The quantity of transcriptionally active RNA Polymerase-II within the Rhodopsin gene (Rho) was significantly increased in the adult neural retina, compared to the immature retina.ConclusionFIZ1 directly interacts with CRX to enhance CRX's transactivation activity for target genes. Developmentally, in neural retina tissue, the increased association of FIZ1 with CRX target genes corresponds to an increased association of transcriptionally active Pol-II within the Rho gene. Together with previous findings, our results suggest that FIZ1 may act as a transcriptional co-regulator of photoreceptor-specific genes, recruited by at least two photoreceptor-specific transcription factors, CRX and NRL. Further studies are underway to elucidate the exact role of FIZ1 in photoreceptor gene expression, development and maintenance.