A single transcription factor is sufficient to induce and maintain secretory cell architecture.

We hypothesized that basic helix-loop-helix (bHLH) MIST1 (BHLHA15) is a "scaling factor" that universally establishes secretory morphology in cells that perform regulated secretion. Here, we show that targeted deletion of MIST1 caused dismantling of the secretory apparatus of diverse exocrine cells. Parietal cells (PCs), whose function is to pump acid into the stomach, normally lack MIST1 and do not perform regulated secretion. Forced expression of MIST1 in PCs caused them to expand their apical cytoplasm, rearrange mitochondrial/lysosome trafficking, and generate large secretory granules. Mist1 induced a cohort of genes regulated by MIST1 in multiple organs but did not affect PC function. MIST1 bound CATATG/CAGCTG E boxes in the first intron of genes that regulate autophagosome/lysosomal degradation, mitochondrial trafficking, and amino acid metabolism. Similar alterations in cell architecture and gene expression were also caused by ectopically inducing MIST1 in vivo in hepatocytes. Thus, MIST1 is a scaling factor necessary and sufficient by itself to induce and maintain secretory cell architecture. Our results indicate that, whereas mature cell types in each organ may have unique developmental origins, cells performing similar physiological functions throughout the body share similar transcription factor-mediated architectural "blueprints."

Singlet Oxygen Leads to Structural Changes to Chloroplasts during their Degradation in the Arabidopsis thaliana plastid ferrochelatase two Mutant.

During stress, chloroplasts produce large amounts of reactive oxygen species (ROS). Chloroplasts also contain many nutrients, including 80% of a leaf's nitrogen supply. Therefore, to protect cells from photo-oxidative damage and to redistribute nutrients to sink tissues, chloroplasts are prime targets for degradation. Multiple chloroplast degradation pathways are induced by photo-oxidative stress or nutrient starvation, but the mechanisms by which damaged or senescing chloroplasts are identified, transported to the central vacuole and degraded are poorly defined. Here, we investigated the structures involved with degrading chloroplasts induced by the ROS singlet oxygen (1O2) in the Arabidopsis thaliana plastid ferrochelatase two (fc2) mutant. Under mild 1O2 stress, most fc2 chloroplasts appeared normal, but had reduced starch content. A subset of chloroplasts was degrading, and some protruded into the central vacuole via 'blebbing' structures. A 3D electron microscopy analysis demonstrated that up to 35% of degrading chloroplasts contained such structures. While the location of a chloroplast within a cell did not affect the likelihood of its degradation, chloroplasts in spongy mesophyll cells were degraded at a higher rate than those in palisade mesophyll cells. To determine if degrading chloroplasts have unique structural characteristics, allowing them to be distinguished from healthy chloroplasts, we analyzed fc2 seedlings grown under different levels of photo-oxidative stress. A correlation was observed among chloroplast swelling, 1O2 signaling and the state of degradation. Finally, plastoglobule (PG) enzymes involved in chloroplast disassembly were upregulated while PGs increased their association with the thylakoid grana, implicating an interaction between 1O2-induced chloroplast degradation and senescence pathways.

Catheterization alters bladder ecology to potentiate Staphylococcus aureus infection of the urinary tract.

Methicillin-resistant Staphylococcus aureus (MRSA) is an emerging cause of catheter-associated urinary tract infection (CAUTI), which frequently progresses to more serious invasive infections. We adapted a mouse model of CAUTI to investigate how catheterization increases an individual's susceptibility to MRSA UTI. This analysis revealed that catheterization was required for MRSA to achieve high-level, persistent infection in the bladder. As shown previously, catheter placement induced an inflammatory response resulting in the release of the host protein fibrinogen (Fg), which coated the bladder and implant. Following infection, we showed that MRSA attached to the urothelium and implant in patterns that colocalized with deposited Fg. Furthermore, MRSA exacerbated the host inflammatory response to stimulate the additional release and accumulation of Fg in the urinary tract, which facilitated MRSA colonization. Consistent with this model, analysis of catheters from patients with S. aureus-positive cultures revealed colocalization of Fg, which was deposited on the catheter, with S. aureus Clumping Factors A and B (ClfA and ClfB) have been shown to contribute to MRSA-Fg interactions in other models of disease. We found that mutants in clfA had significantly greater Fg-binding defects than mutants in clfB in several in vitro assays. Paradoxically, only the ClfB- strain was significantly attenuated in the CAUTI model. Together, these data suggest that catheterization alters the urinary tract environment to promote MRSA CAUTI pathogenesis by inducing the release of Fg, which the pathogen enhances to persist in the urinary tract despite the host's robust immune response.

Reciprocal Spatiotemporally Controlled Apoptosis Regulates Wolffian Duct Cloaca Fusion.

The epithelial Wolffian duct (WD) inserts into the cloaca (primitive bladder) before metanephric kidney development, thereby establishing the initial plumbing for eventual joining of the ureters and bladder. Defects in this process cause common anomalies in the spectrum of congenital anomalies of the kidney and urinary tract (CAKUT). However, developmental, cellular, and molecular mechanisms of WD-cloaca fusion are poorly understood. Through systematic analysis of early WD tip development in mice, we discovered that a novel process of spatiotemporally regulated apoptosis in WD and cloaca was necessary for WD-cloaca fusion. Aberrant RET tyrosine kinase signaling through tyrosine (Y) 1062, to which PI3K- or ERK-activating proteins dock, or Y1015, to which PLCγ docks, has been shown to cause CAKUT-like defects. Cloacal apoptosis did not occur in RetY1062F mutants, in which WDs did not reach the cloaca, or in RetY1015F mutants, in which WD tips reached the cloaca but did not fuse. Moreover, inhibition of ERK or apoptosis prevented WD-cloaca fusion in cultures, and WD-specific genetic deletion of YAP attenuated cloacal apoptosis and WD-cloacal fusion in vivo Thus, cloacal apoptosis requires direct contact and signals from the WD tip and is necessary for WD-cloacal fusion. These findings may explain the mechanisms of many CAKUT.

In vitro myelin formation using embryonic stem cells.

Myelination in the central nervous system is the process by which oligodendrocytes form myelin sheaths around the axons of neurons. Myelination enables neurons to transmit information more quickly and more efficiently and allows for more complex brain functions; yet, remarkably, the underlying mechanism by which myelination occurs is still not fully understood. A reliable in vitro assay is essential to dissect oligodendrocyte and myelin biology. Hence, we developed a protocol to generate myelinating oligodendrocytes from mouse embryonic stem cells and established a myelin formation assay with embryonic stem cell-derived neurons in microfluidic devices. Myelin formation was quantified using a custom semi-automated method that is suitable for larger scale analysis. Finally, early myelination was followed in real time over several days and the results have led us to propose a new model for myelin formation.

Characterization of a Mycobacterium tuberculosis nanocompartment and its potential cargo proteins.

Mycobacterium tuberculosis has evolved various mechanisms by which the bacterium can maintain homeostasis under numerous environmental assaults generated by the host immune response. M. tuberculosis harbors enzymes involved in the oxidative stress response that aid in survival during the production of reactive oxygen species in activated macrophages. Previous studies have shown that a dye-decolorizing peroxidase (DyP) is encapsulated by a bacterial nanocompartment, encapsulin (Enc), whereby packaged DyP interacts with Enc via a unique C-terminal extension. M. tuberculosis also harbors an encapsulin homolog (CFP-29, Mt-Enc), within an operon with M. tuberculosis DyP (Mt-DyP), which contains a C-terminal extension. Together these observations suggest that Mt-DyP interacts with Mt-Enc. Furthermore, it has been suggested that DyPs may function as either a heme-dependent peroxidase or a deferrochelatase. Like Mt-DyP, M. tuberculosis iron storage ferritin protein, Mt-BfrB, and an M. tuberculosis protein involved in folate biosynthesis, 7,8-dihydroneopterin aldolase (Mt-FolB), have C-terminal tails that could also interact with Mt-Enc. For the first time, we show by co-purification and electron microscopy that mycobacteria via Mt-Enc can encapsulate Mt-DyP, Mt-BfrB, and Mt-FolB. Functional studies of free or encapsulated proteins demonstrate that they retain their enzymatic activity within the Mt-Enc nanocompartment. Mt-DyP, Mt-FolB, and Mt-BfrB all have antioxidant properties, suggesting that if these proteins are encapsulated by Mt-Enc, then this nanocage may play a role in the M. tuberculosis oxidative stress response. This report provides initial structural and biochemical clues regarding the molecular mechanisms that utilize compartmentalization by which the mycobacterial cell may aid in detoxification of the local environment to ensure long term survival.

Maturation of Heterogeneity in Afferent Synapse Ultrastructure in the Mouse Cochlea.

Auditory nerve fibers (ANFs) innervating the same inner hair cell (IHC) may have identical frequency tuning but different sound response properties. In cat and guinea pig, ANF response properties correlate with afferent synapse morphology and position on the IHC, suggesting a causal structure-function relationship. In mice, this relationship has not been fully characterized. Here we measured the emergence of synaptic morphological heterogeneities during maturation of the C57BL/6J mouse cochlea by comparing postnatal day 17 (p17, ∼3 days after hearing onset) with p34, when the mouse cochlea is mature. Using serial block face scanning electron microscopy and three-dimensional reconstruction we measured the size, shape, vesicle content, and position of 70 ribbon synapses from the mid-cochlea. Several features matured over late postnatal development. From p17 to p34, presynaptic densities (PDs) and post-synaptic densities (PSDs) became smaller on average (PDs: 0.75 to 0.33; PSDs: 0.58 to 0.31 µm2) and less round as their short axes shortened predominantly on the modiolar side, from 770 to 360 nm. Membrane-associated synaptic vesicles decreased in number from 53 to 30 per synapse from p17 to p34. Anatomical coupling, measured as PSD to ribbon distance, tightened predominantly on the pillar side. Ribbons became less spherical as long-axes lengthened only on the modiolar side of the IHC, from 372 to 541 nm. A decreasing gradient of synaptic ribbon size along the modiolar-pillar axis was detected only at p34 after aligning synapses of adjacent IHCs to a common reference frame (median volumes in nm3 × 106: modiolar 4.87; pillar 2.38). The number of ribbon-associated synaptic vesicles scaled with ribbon size (range 67 to 346 per synapse at p34), thus acquiring a modiolar-pillar gradient at p34, but overall medians were similar at p17 (120) and p34 (127), like ribbon surface area (0.36 vs. 0.34 µm2). PD and PSD morphologies were tightly correlated to each other at individual synapses, more so at p34 than p17, but not to ribbon morphology. These observations suggest that PDs and PSDs mature according to different cues than ribbons, and that ribbon size may be more influenced by cues from the IHC than the surrounding tissue.

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice.

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of UTI in male and female mice have illustrated the procedures for bacterial inoculation and enumeration in urine and tissues. During an initial bladder infection in C57BL/6 mice, UPEC establish latent reservoirs inside bladder epithelial cells that persist following clearance of UPEC bacteriuria. This model builds on these studies to examine rUTI caused by the emergence of UPEC from within latent bladder reservoirs. The urogenital bacterium Gardnerella vaginalis is used as the trigger of rUTI in this model because it is frequently present in the urogenital tracts of women, especially in the context of vaginal dysbiosis that has been associated with UTI. In addition, a method for in situ bladder fixation followed by scanning electron microscopy (SEM) analysis of bladder tissue is also described, with potential application to other studies involving the bladder.

Characterization of the bone marrow adipocyte niche with three-dimensional electron microscopy.

Unlike white and brown adipose tissues, the bone marrow adipocyte (BMA) exists in a microenvironment containing unique populations of hematopoietic and skeletal cells. To study this microenvironment at the sub-cellular level, we performed a three-dimensional analysis of the ultrastructure of the BMA niche with focused ion beam scanning electron microscopy (FIB-SEM). This revealed that BMAs display hallmarks of metabolically active cells including polarized lipid deposits, a dense mitochondrial network, and areas of endoplasmic reticulum. The distinct orientations of the triacylglycerol droplets suggest that fatty acids are taken up and/or released in three key areas - at the endothelial interface, into the hematopoietic milieu, and at the bone surface. Near the sinusoidal vasculature, endothelial cells send finger-like projections into the surface of the BMA which terminate near regions of lipid within the BMA cytoplasm. In some regions, perivascular cells encase the BMA with their flattened cellular projections, limiting contacts with other cells in the niche. In the hematopoietic milieu, BMAT adipocytes of the proximal tibia interact extensively with maturing cells of the myeloid/granulocyte lineage. Associations with erythroblast islands are also prominent. At the bone surface, the BMA extends organelle and lipid-rich cytoplasmic regions toward areas of active osteoblasts. This suggests that the BMA may serve to partition nutrient utilization between diverse cellular compartments, serving as an energy-rich hub of the stromal-reticular network. Lastly, though immuno-EM, we've identified a subset of bone marrow adipocytes that are innervated by the sympathetic nervous system, providing an additional mechanism for regulation of the BMA. In summary, this work reveals that the bone marrow adipocyte is a dynamic cell with substantial capacity for interactions with the diverse components of its surrounding microenvironment. These local interactions likely contribute to its unique regulation relative to peripheral adipose tissues.

Modern Laser Scanning Confocal Microscopy.

Since its commercialization in the late 1980's, confocal laser scanning microscopy (CLSM) has since become one of the most prevalent fluorescence microscopy techniques for three-dimensional structural studies of biological cells and tissues. The flexibility of the approach has enabled its application in a diverse array of studies, from the fast imaging of dynamic processes in living cells, to meticulous morphological analyses of tissues, and co-localization of protein expression patterns. In this chapter, we introduce the principles of confocal microscopy and discuss how the approach has become a mainstay in the biological sciences. We describe the components of a CLSM system and assess how modern implementations of the approach have further expanded the use of the technique. Finally, we briefly outline some practical considerations to take into account when acquiring data using a CLSM system. © 2018 by John Wiley & Sons, Inc.

Nonlinear optical imaging of extracellular matrix proteins.

Over the last 2 decades, nonlinear imaging methods such as multiharmonic imaging microscopy (MHIM) have become powerful approaches for the label-free visualization of biological structures. Multiharmonic signals are generated when an intense electromagnetic field propagates through a sample that either has a specific molecular orientation or exhibits certain physical properties. It can provide complementary morphological information when integrated with other nonlinear optical imaging techniques such as two-photon excitation (TPE). Here, we present the necessary methodology to implement an integrated approach for multiharmonic and TPE imaging of the mouse aorta using a commercial two-photon microscope. This approach illustrates how to differentiate the microstructure of the mouse aorta that are due to collagen fibrils and elastic laminae under 820 and 1230nm excitation. Our method also demonstrates how to perform multiharmonic generation by reflectance of the forwardly propagating emission signal. The ability to visualize biological samples without additional genetically targeted or chemical stains makes MHIM well suited for studying the morphology of the mouse aorta and has the potential to be applied to other collagen and elastin-rich tissues.

Red blood cell phenotype fidelity following glycerol cryopreservation optimized for research purposes.

Intact red blood cells (RBCs) are required for phenotypic analyses. In order to allow separation (time and location) between subject encounter and sample analysis, we developed a research-specific RBC cryopreservation protocol and assessed its impact on data fidelity for key biochemical and physiological assays. RBCs drawn from healthy volunteers were aliquotted for immediate analysis or following glycerol-based cryopreservation, thawing, and deglycerolization. RBC phenotype was assessed by (1) scanning electron microscopy (SEM) imaging and standard morphometric RBC indices, (2) osmotic fragility, (3) deformability, (4) endothelial adhesion, (5) oxygen (O2) affinity, (6) ability to regulate hypoxic vasodilation, (7) nitric oxide (NO) content, (8) metabolomic phenotyping (at steady state, tracing with [1,2,3-13C3]glucose ± oxidative challenge with superoxide thermal source; SOTS-1), as well as in vivo quantification (following human to mouse RBC xenotransfusion) of (9) blood oxygenation content mapping and flow dynamics (velocity and adhesion). Our revised glycerolization protocol (40% v/v final) resulted in >98.5% RBC recovery following freezing (-80°C) and thawing (37°C), with no difference compared to the standard reported method (40% w/v final). Full deglycerolization (>99.9% glycerol removal) of 40% v/v final samples resulted in total cumulative lysis of ~8%, compared to ~12-15% with the standard method. The post cryopreservation/deglycerolization RBC phenotype was indistinguishable from that for fresh RBCs with regard to physical RBC parameters (morphology, volume, and density), osmotic fragility, deformability, endothelial adhesivity, O2 affinity, vasoregulation, metabolomics, and flow dynamics. These results indicate that RBC cryopreservation/deglycerolization in 40% v/v glycerol final does not significantly impact RBC phenotype (compared to fresh cells).

HID-1 controls formation of large dense core vesicles by influencing cargo sorting and trans-Golgi network acidification.

Large dense core vesicles (LDCVs) mediate the regulated release of neuropeptides and peptide hormones. They form at the trans-Golgi network (TGN), where their soluble content aggregates to form a dense core, but the mechanisms controlling biogenesis are still not completely understood. Recent studies have implicated the peripheral membrane protein HID-1 in neuropeptide sorting and insulin secretion. Using CRISPR/Cas9, we generated HID-1 KO rat neuroendocrine cells, and we show that the absence of HID-1 results in specific defects in peptide hormone and monoamine storage and regulated secretion. Loss of HID-1 causes a reduction in the number of LDCVs and affects their morphology and biochemical properties, due to impaired cargo sorting and dense core formation. HID-1 KO cells also exhibit defects in TGN acidification together with mislocalization of the Golgi-enriched vacuolar H+-ATPase subunit isoform a2. We propose that HID-1 influences early steps in LDCV formation by controlling dense core formation at the TGN.

Light Sheet Fluorescence Microscopy (LSFM).

The development of confocal microscopy techniques introduced the ability to optically section fluorescent samples in the axial dimension, perpendicular to the image plane. These approaches, via the placement of a pinhole in the conjugate image plane, provided superior resolution in the axial (z) dimension resulting in nearly isotropic optical sections. However, increased axial resolution, via pinhole optics, comes at the cost of both speed and excitation efficiency. Light sheet fluorescent microscopy (LSFM), a century-old idea made possible with modern developments in both excitation and detection optics, provides sub-cellular resolution and optical sectioning capabilities without compromising speed or excitation efficiency. Over the past decade, several variations of LSFM have been implemented each with its own benefits and deficiencies. Here we discuss LSFM fundamentals and outline the basic principles of several major light-sheet-based imaging modalities (SPIM, inverted SPIM, multi-view SPIM, Bessel beam SPIM, and stimulated emission depletion SPIM) while considering their biological relevance in terms of intrusiveness, temporal resolution, and sample requirements.

Ubiquitin facilitates a quality-control pathway that removes damaged chloroplasts.

Energy production by chloroplasts and mitochondria causes constant oxidative damage. A functioning photosynthetic cell requires quality-control mechanisms to turn over and degrade chloroplasts damaged by reactive oxygen species (ROS). Here, we generated a conditionally lethal Arabidopsis mutant that accumulated excess protoporphyrin IX in the chloroplast and produced singlet oxygen. Damaged chloroplasts were subsequently ubiquitinated and selectively degraded. A genetic screen identified the plant U-box 4 (PUB4) E3 ubiquitin ligase as being necessary for this process. pub4-6 mutants had defects in stress adaptation and longevity. Thus, we have identified a signal that leads to the targeted removal of ROS-overproducing chloroplasts.

Helium Ion Microscopy (HIM) for the imaging of biological samples at sub-nanometer resolution.

Scanning Electron Microscopy (SEM) has long been the standard in imaging the sub-micrometer surface ultrastructure of both hard and soft materials. In the case of biological samples, it has provided great insights into their physical architecture. However, three of the fundamental challenges in the SEM imaging of soft materials are that of limited imaging resolution at high magnification, charging caused by the insulating properties of most biological samples and the loss of subtle surface features by heavy metal coating. These challenges have recently been overcome with the development of the Helium Ion Microscope (HIM), which boasts advances in charge reduction, minimized sample damage, high surface contrast without the need for metal coating, increased depth of field, and 5 angstrom imaging resolution. We demonstrate the advantages of HIM for imaging biological surfaces as well as compare and contrast the effects of sample preparation techniques and their consequences on sub-nanometer ultrastructure.

Super-resolution microscopy: a comparative treatment.

One of the fundamental limitations of optical microscopy is that of diffraction, or in essence, how small a beam of light can be focused by using an optical lens system. This constraint, or barrier if you will, was theoretically described by Ernst Abbe in 1873 and is roughly equal to half the wavelength of light used to probe the system. Many structures, particularly those within cells, are much smaller than this limit and thus are difficult to visualize. Over the last two decades, a new field of super-resolution imaging has been created and been developed into a broad range of techniques that allow routine imaging beyond the far-field diffraction limit of light. In this unit we outline the basic principles of the various super-resolution imaging modalities, paying particular attention to the technical considerations for biological imaging. Furthermore, we discuss their various applications in the imaging of both fixed and live biological samples.