43 research outputs found
INVESTIGATING THE MECHANISM OF BACTERIAL CELL DIVISION WITH SUPERRESOLUTION MICROSCOPY
No full textThe molecular mechanisms that drive bacterial cytokinesis are attractive antibiotic targets that remain poorly understood. The machinery that performs cytokinesis in bacteria has been termed the 'divisome' (see Chapter 1 for description). The most widely-conserved divisome protein, FtsZ, is an essential tubulin homolog that polymerizes into protofilaments in a nucleotide-dependent manner. These protofilaments assemble at midcell to form the ‘Z-ring’, which has been the prevailing candidate for constrictive force generation during cell division. However, it has been difficult to experimentally test proposed Z-ring force generation models in vivo due to the small size of bacteria (< 1 μm diameter for E. coli) compared to the diffraction-limited resolution of light (~ 0.3 μm).
In this work, quantitative superresolution and time-lapse microscopy were applied to examine whether Z-ring structure and function indeed play limiting roles in driving E. coli cell constriction (Chapter 2). Surprisingly, these studies revealed that the rate of septum closure during constriction is robust to substantial changes in many Z-ring properties, including the GTPase activity of FtsZ, molecular density of the Z-ring, the timing of Z-ring disassembly, and the absence of Z-ring assembly regulators. Further investigation revealed that septum closure rate is instead highly coupled to the rate of cell wall growth and elongation, and can be modulated by coordination with chromosome segregation. Taken together, these results challenge the Z-ring centric view of constriction force generation, and suggest that cell wall synthesis and chromosome segregation likely drive the rate and progress of cell constriction in bacteria.
These investigations were made possible by advancements in quantitative superresolution microscopy techniques (see Chapter 3 for overview). One major obstacle encountered during the course of this work, and shared by those utilizing localization-based superresolution microscopy techniques, was the overestimation of molecule numbers caused by fluorophore photoblinking. Thus, Chapter 4 describes a systematic characterization of the effects of photoblinking on the accurate construction and analysis of superresolution images. These characterizations enabled the development of a simple method to identify the optimal clustering thresholds and an empirical criterion to evaluate whether an imaging condition is appropriate for accurate superresolution image reconstruction. Both the threshold selection method and imaging condition criterion are easy to implement within existing PALM clustering algorithms and experimental conditions
Abstract 3832: Optimization strategy for fluorescent multiplex immunohistochemistry tissue staining
No full textAbstract
Introduction: Recent insights into the tumor microenvironment have fueled the need for additional information beyond the one or two phenotypes provided by traditional immunohistochemistry (IHC). As a response, manual and automated fluorescent multiplex immunohistochemistry (fIHC) techniques have been recently developed and accepted by the immuno-oncology space. Fluorescent multiplex immunohistochemistry (fIHC) assays are designed to simultaneously measure multiple biomarkers in tissue sections with visual context that is lost in other methods, such as flow cytometry. Here we describe a novel optimization strategy to achieve quantitative, robust, and specific multiplex fIHC staining results with both manual and automated multiple-color Opal procedures.
Methods: Formalin-fixed paraffin-embedded samples of primary tumors were immunostained using Opal™ reagents both manually and on a fully automated Leica BOND RX™ stainer. The impact of different reagent concentrations and quantities were analyzed in respect to signal specificity and robustness, stripping efficiency, signal co-localization, signal to noise, and color separation. Images were acquired on a Vectra 3.0® automated imaging system, and analyzed with inForm® software.
Results: The goals of this study were two-fold:
-to understand the impact of Opal reagent concentrations/quantities on fluorescent signal intensities acquired from cells within the context of tissue.
-to develop an Opal reagent optimization method that yields more consistent, quantitative results from separated and co-localized fluorescent signals.
We’ve applied this novel fIHC experimental approach and optimization strategy to Opal monoplex and multiplex assays and explored staining robustness, contextual specificity, staining order independence, and co-localized signal separation. Using the novel optimization strategy, we have achieved optimal staining patterns with improved confidence in the quantitative characteristics of the assay. Tissue sections stained after optimization have exhibited staining order independence and closely align with traditional IHC patterns.
Conclusion: This novel optimization strategy, developed for Opal fIHC assays, is more quantitative, improves staining results, and minimizes interference between co-localized biomarkers.
Citation Format: Yi zheng, Carla Coltharp, Ryan Dilworth, Linying Liu, Darryn Unfricht, Cliff Hoyt, Milind Rajopadhye, Peter Miller. Optimization strategy for fluorescent multiplex immunohistochemistry tissue staining [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3832. doi:10.1158/1538-7445.AM2017-3832</jats:p
Beyond force generation: Why is a dynamic ring of FtsZ polymers essential for bacterial cytokinesis?
No full textStructures and Dynamics of the E. Coli FtsZ-Ring and its Associated Proteins during Cell Division Revealed by Superresolution Imaging
No full textSuperresolution Investigation of the Dynamics of FtsZ Structures during E. Coli Cell Division
No full textAccurate construction of photoactivated localization microscopy (PALM) images for quantitative measurements.
No full textLocalization-based superresolution microscopy techniques such as Photoactivated Localization Microscopy (PALM) and Stochastic Optical Reconstruction Microscopy (STORM) have allowed investigations of cellular structures with unprecedented optical resolutions. One major obstacle to interpreting superresolution images, however, is the overcounting of molecule numbers caused by fluorophore photoblinking. Using both experimental and simulated images, we determined the effects of photoblinking on the accurate reconstruction of superresolution images and on quantitative measurements of structural dimension and molecule density made from those images. We found that structural dimension and relative density measurements can be made reliably from images that contain photoblinking-related overcounting, but accurate absolute density measurements, and consequently faithful representations of molecule counts and positions in cellular structures, require the application of a clustering algorithm to group localizations that originate from the same molecule. We analyzed how applying a simple algorithm with different clustering thresholds (t(Thresh) and d(Thresh)) affects the accuracy of reconstructed images, and developed an easy method to select optimal thresholds. We also identified an empirical criterion to evaluate whether an imaging condition is appropriate for accurate superresolution image reconstruction with the clustering algorithm. Both the threshold selection method and imaging condition criterion are easy to implement within existing PALM clustering algorithms and experimental conditions. The main advantage of our method is that it generates a superresolution image and molecule position list that faithfully represents molecule counts and positions within a cellular structure, rather than only summarizing structural properties into ensemble parameters. This feature makes it particularly useful for cellular structures of heterogeneous densities and irregular geometries, and allows a variety of quantitative measurements tailored to specific needs of different biological systems
Direct Activation of Epac by Sulfonylurea Is Isoform Selective
No full textSummaryCommonly used as a treatment for Type II diabetes, sulfonylureas (SUs) stimulate insulin secretion from pancreatic β cells by binding to sulfonylurea receptors. Recently, SUs have been shown to also activate exchange protein directly activated by cAMP 2 (Epac2), however, little is known about this molecular action. Using biosensor imaging and biochemical analysis, we show that SUs activate Epac2 and the downstream signaling via direct binding to Epac2. We further identify R447 of Epac2 to be critically involved in SU binding. This distinct binding site from cAMP points to a new mode of allosteric activation of Epac2. We also show that SUs selectively activate Epac2 isoform, but not the closely related Epac1, further establishing SUs as a new class of isoform-selective enzyme activators
A Super-Resolution Study of the Structural Organization of the E. Coli FTSZ-Ring by ZapA and ZapB
No full textAccuracy of images generated with different threshold pairs.
No full text<p>(A) Region of threshold space (white squares) that resulted in <10% difference from the reference measurements of Z-ring width, <i>N</i>, and <i>f<sub>midcell</sub></i>, and that yielded Z-ring density distributions not significantly different from the reference distribution (pKS >0.05). (B) Jaccard index values at each threshold pair. Higher Jaccard index values indicate more accurate single-molecule clustering. (C) The peak of the Jaccard index plot (B, white squares) is within the region where all four quantitative measurements are within 10% of the reference measurements (A). Dataset analyzed is the same simulated dataset shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051725#pone-0051725-g002" target="_blank">Figure 2</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051725#pone-0051725-g004" target="_blank">4</a>.</p
