Breaching the Barrier: Quantifying Antibiotic Permeability across Gram-negative Bacterial Membranes.

The double-membrane cell envelope of Gram-negative bacteria is a sophisticated barrier that facilitates the uptake of nutrients and protects the organism from toxic compounds. An antibiotic molecule must find its way through the negatively charged lipopolysaccharide layer on the outer surface, pass through either a porin or the hydrophobic layer of the outer membrane, then traverse the hydrophilic peptidoglycan layer only to find another hydrophobic lipid bilayer before it finally enters the cytoplasm, where it typically finds its target. This complex uptake pathway with very different physico-chemical properties is one reason that Gram-negative are intrinsically protected against multiple classes of antibiotic-like molecules, and is likely the main reason that in vitro target-based screening programs have failed to deliver novel antibiotics for these organisms. Due to the lack of general methods available for quantifying the flux of drugs into the cell, little is known about permeation rates, transport pathways and accumulation at the target sites for particular molecules. Here we summarize the current tools available for measuring antibiotic uptake across the different compartments of Gram-negative bacteria

Germinal center B cells recognize antigen through a specialized immune synapse architecture

B cell activation is regulated by B cell antigen receptor (BCR) signaling and antigen internalization in immune synapses. Using large-scale imaging across B cell subsets, we show that in contrast to naive and memory B cells, which gathered antigen towards the synapse center before internalization, germinal center (GC) B cells extracted antigen by a distinct pathway using small peripheral clusters. Both naive and GC B cell synapses required proximal BCR signaling, but GC cells signaled less through the protein kinase C-β (PKC-β)–NF-κB pathway and produced stronger tugging forces on the BCR, thereby more stringently regulating antigen binding. Consequently, GC B cells extracted antigen with better affinity discrimination than naive B cells, suggesting that specialized biomechanical patterns in B cell synapses regulate T-cell dependent selection of high-affinity B cells in GCs

Redox signals at the ER-mitochondria interface control melanoma progression.

Reactive oxygen species (ROS) are emerging as important regulators of cancer growth and metastatic spread. However, how cells integrate redox signals to affect cancer progression is not fully understood. Mitochondria are cellular redox hubs, which are highly regulated by interactions with neighboring organelles. Here, we investigated how ROS at the endoplasmic reticulum (ER)-mitochondria interface are generated and translated to affect melanoma outcome. We show that TMX1 and TMX3 oxidoreductases, which promote ER-mitochondria communication, are upregulated in melanoma cells and patient samples. TMX knockdown altered mitochondrial organization, enhanced bioenergetics, and elevated mitochondrial- and NOX4-derived ROS. The TMX-knockdown-induced oxidative stress suppressed melanoma proliferation, migration, and xenograft tumor growth by inhibiting NFAT1. Furthermore, we identified NFAT1-positive and NFAT1-negative melanoma subgroups, wherein NFAT1 expression correlates with melanoma stage and metastatic potential. Integrative bioinformatics revealed that genes coding for mitochondrial- and redox-related proteins are under NFAT1 control and indicated that TMX1, TMX3, and NFAT1 are associated with poor disease outcome. Our study unravels a novel redox-controlled ER-mitochondria-NFAT1 signaling loop that regulates melanoma pathobiology and provides biomarkers indicative of aggressive disease

3D imaging and quantitative analysis of intact tissues and organs

Embryonic development and tumor growth are highly complex and dynamic processes that exist in both time and space. To fully understand the molecular mechanisms that control these processes, it is crucial to study RNA expression and protein translation with single-cell spatiotemporal resolution. This is feasible by microscopic imaging that enables multidimensional assessments of cells, tissues, and organs. Here, a time-lapse calcium imaging and three-dimensional imaging was used to study physiological development of the brain or pathological development of cancer, respectively. In Paper I, spatiotemporal calcium imaging revealed a new mechanism of neurogenesis during brain development. In Paper II, a new clearing method of clinically stored specimens, DIPCO (diagnosing immunolabeled paraffin-embedded cleared organs), was developed that allows better characterization and staging of intact human tumors. In Paper III, the DIPCO method was applied to determine tumor stage and characterize the microlymphatic system in bladder cancer. In Paper IV, a novel method for RNA labeling of volumetric specimens, DIIFCO (diagnosing in situ and immunofluorescence-labeled cleared onco-sample) was developed to study RNAs expression and localization in intact tumors. Overall, the aim of the thesis was to demonstrate that multidimensional imaging extends the understanding of both physiological and pathological biological developmental processes

Quantifying Non-Muscle Myosin 2 Filament Dynamics

Non-muscle myosin 2 is a motor protein that, when assembled into filaments, works on the actin cytoskeleton to produce contractile forces within cells. This type of myosin dynamically assembles within cells to exert forces necessary for a wide variety of functions including cell division, migration, and multicellular movement. The ability to dynamically assemble contractile networks, and the ability to control the assembly and placement of these myosin filaments in both space and time, is therefore required throughout cell physiology. We used a suite of dynamic, quantitative imaging approaches to identify deterministic factors that drive myosin filament appearance and amplification as well as characterize novel movement of assembled myosin filaments. We found that actin dynamics regulate myosin assembly - particularly that remodeling of actin networks, not architecture, modulates the local myosin monomer levels and facilitates assembly through myosin:myosin driven interactions. Using optogenetically controlled myosin, we demonstrate that locally concentrating myosin is sufficient to both form filaments and jump-start filament amplification and partitioning. By counting myosin monomers within filaments, we demonstrate a myosin-facilitated assembly process that establishes sub-resolution filament stacks prior to partitioning into clusters that feed higher-order networks. Additionally, we report novel in vivo observations of processive myosin filament movements in cells, similar to known cargo trafficking motors. Together these findings establish biophysical mechanisms regulating the assembly of non-muscle contractile structures that are ubiquitous throughout cell biology. This work also invites future research into mechanisms of myosin monomer crowding to drive contractility during myriad biological processes

Aqueous Two-Phase System Patterning for Crosstalk-free Multiplexed Detection of Plasma Biomarkers.

Highly sensitive and robust protein assay platforms are urgently needed to improve diagnosis and inform therapeutic decisions in many disease areas. However, current bioassays for multiplexed protein biomarker detection are plagued by nonspecific antibody cross-reactions, which often lead to false positive detection and misdiagnoses. The work presented in this dissertation fills this key technological gap in protein assay development, and describes two innovative multiplexed bioassays that completely eliminate antibody cross-reactions. First, we introduce a multiplexed bead-based assay that not only prevents antibody cross-reactions, but also does not require any wash steps. We circumvented the antibody crosstalk problem by stably co-localizing antibody-conjugated beads in droplets of immiscible aqueous polymer solutions. We demonstrated this assay’s clinical utility by measuring the levels of proinflammatory biomarkers (chemokine ligand 9, chemokine ligand 10, interleukin-6, and interleuikin-8) in plasma samples from from 88 bone marrow transplant (BMT) patients. Our assay accurately discriminated BMT patients afflicted with and without chronic graft-versus-host disease (GVHD) patients within 2 hours. Second, we describe a multiplexed ELISA that eradicates antibody cross-reactions by confining and spatially patterning detection antibodies within droplets of phase-separating polymers. We used this crosstalk-free assay to measure a panel of four acute GVHD biomarkers (tumor necrosis factor-alpha, hepatocyte growth factor, elafin, and ST2) in plasma samples from 81 BMT patients. Not only does this assay eliminate antibody crosstalk, but it also reduces antibody costs by 100-fold because assays only require 0.5-microliters detection antibody volume instead of 50-microliters required by conventional ELISA. Notably, both crosstalk-free multiplexed platforms can be read using commercially available plate readers, indicating that these assay platforms can be valuable tools for diagnosis complex diseases like GVHD.PHDMacromolecular Science & EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/99822/1/arlynes_1.pd

New Methodologies for Studying Intercellular Connectivity and Signaling in Plant Tissues

Cells are at the core of our biosphere. Their organization into tissues, organs, organ systems, and organisms requires frequent exchange of nutrients and information to promote proper developmental patterning and organismal success. Direct physical connections that unite the cytoplasm of adjacent cells provide the most energy-efficient mechanism for moving substances from one cell to the next. Therefore, it is important to understand both the subcellular mechanisms that govern cell-to-cell transport and the supracellular processes that utilize these physical connections. In a plant organism, intercellular communication is dictated by the numerous pores in a plant cell wall that enable the unity of cytoplasm between adjacent cells within and often between tissues. These so-called plasmodesmata are vital in plant physiology at the supracellular level. As plants cannot escape either biotic or abiotic stressors, they have developed sophisticated chemical signaling processes that utilize plasmodesmata to coordinate a response, if necessary.The microinjection of fluorescent probes into live cells is an essential component in the toolbox of modern plant cell biology because it allows for a direct assessment of plasmodesmal function. However, traditional methods to introduce fluorescent probes into living cells necessarily introduce artifacts that limit our progress. Herein, I describe the diffusive injection micropipette (DIMP) which was developed to avoid several of the artifacts that impact cell function during traditional injections. I used DIMPs to successfully introduce fluorescent probes into plant, fungal, and animal cells, with a more in-depth investigation into the function of plant cell plasmodesmata. As plasmodesmata are nanoscopic channels with complex anatomical features, they are subject to certain biophysical processes that are absent at the macroscopic level. At the nanoscopic scale, surface charges of the plasma membrane generate an electric field that may be large enough to interact with molecules passing through plasmodesmata. Using the DIMP, I found that positively charged fluorescent probes were greatly impaired in their cell-to-cell movement via plasmodesmata when compared to negatively charged probes. This is intriguing because calcium signaling cascades that are vital to plant stress responses previously had been hypothesized to involve the movement of calcium through plasmodesmata. Therefore, I also investigated the propagation of calcium signals in local cell populations responding to mechanical stimuli. In response to mechanical stimuli that increase cell turgor, calcium signals depended on plasmodesmata for their propagation within the tissue. However, the signaling cascade following turgor decreases were independent of plasmodesmata and likely utilize extracellular pathways

Advanced optical imaging for the rational design of nanomedicines

Despite the enormous potential of nanomedicines to shape the future of medicine, their clinical translation remains suboptimal. Translational challenges are present in every step of the development pipeline, from a lack of understanding of patient heterogeneity to insufficient insights on nanoparticle properties and their impact on material-cell interactions. Here, we discuss how the adoption of advanced optical microscopy techniques, such as super-resolution optical microscopies, correlative techniques, and high-content modalities, could aid the rational design of nanocarriers, by characterizing the cell, the nanomaterial, and their interaction with unprecedented spatial and/or temporal detail. In this nanomedicine arena, we will discuss how the implementation of these techniques, with their versatility and specificity, can yield high volumes of multi-parametric data; and how machine learning can aid the rapid advances in microscopy: from image acquisition to data interpretation.</p