Directed evolution of the multicopper oxidase laccase for cell surface proximity labeling and electron microscopy.

Enzymes that oxidize aromatic substrates have shown utility in a range of cell-based technologies including live cell proximity labeling (PL) and electron microscopy (EM), but are associated with drawbacks such as the need for toxic HO. Here, we explore laccases as a novel enzyme class for PL and EM in mammalian cells. LaccID, generated via 11 rounds of directed evolution from an ancestral fungal laccase, catalyzes the one-electron oxidation of diverse aromatic substrates using O instead of toxic HO, and exhibits activity selective to the surface plasma membrane of both living and fixed cells.

New rabies viral resources for multi-scale neural circuit mapping.

Comparisons and linkage between multiple imaging scales are essential for neural circuit connectomics. Here, we report 20 new recombinant rabies virus (RV) vectors that we have developed for multi-scale and multi-modal neural circuit mapping tools. Our new RV tools for mesoscale imaging express a range of improved fluorescent proteins.

Fe-TAMLs as a new class of small molecule peroxidase probes for correlated light and electron microscopy.

We introduce Fe-TAML, a small molecule-based peroxidase as a versatile new member of the correlated fluorescence and electron microscopy toolkit. The utility of the probe is demonstrated by high resolution imaging of newly synthesized DNA (through biorthogonal labeling), genetically tagged proteins (using HaloTag), and untagged endogenous proteins (via immunostaining). EM visualization in these applications is facilitated by exploiting Fe-TAML's catalytic activity for the deposition of localized osmiophilic precipitates based on polymerized 3,3'-diaminobenzidine.

Elemental mapping of labelled biological specimens at intermediate energy loss in an energy-filtered TEM acquired using a direct detection device.

The technique of colour EM that was recently developed enabled localisation of specific macromolecules/proteins of interest by the targeted deposition of diaminobenzidine (DAB) conjugated to lanthanide chelates. By acquiring lanthanide elemental maps by energy-filtered transmission electron microscopy (EFTEM) and overlaying them in pseudo-colour over the conventional greyscale TEM image, a colour EM image is generated. This provides a powerful tool for visualising subcellular component/s, by the ability to clearly distinguish them from the general staining of the endogenous cellular material.

Iron-specific Signal Separation from within Heavy Metal Stained Biological Samples Using X-Ray Microtomography with Polychromatic Source and Energy-Integrating Detectors.

Biological samples are frequently stained with heavy metals in preparation for examining the macro, micro and ultra-structure using X-ray microtomography and electron microscopy. A single X-ray microtomography scan reveals detailed 3D structure based on staining density, yet it lacks both material composition and functional information. Using a commercially available polychromatic X-ray source, energy integrating detectors and a two-scan configuration labelled by their energy- "High" and "Low", we demonstrate how a specific element, here shown with iron, can be detected from a mixture with other heavy metals.

Multicolor Electron Microscopy for Simultaneous Visualization of Multiple Molecular Species.

Electron microscopy (EM) remains the primary method for imaging cellular and tissue ultrastructure, although simultaneous localization of multiple specific molecules continues to be a challenge for EM. We present a method for obtaining multicolor EM views of multiple subcellular components. The method uses sequential, localized deposition of different lanthanides by photosensitizers, small-molecule probes, or peroxidases.

PKMζ, but not PKCλ, is rapidly synthesized and degraded at the neuronal synapse.

Synthesizing, localizing, and stabilizing new protein copies at synapses are crucial factors in maintaining the synaptic changes required for storing long-term memories. PKMζ recently emerged as a molecule putatively responsible for maintaining encoded memories over time because its presence correlates with late LTP and because its inhibition disrupts LTP in vitro and long-term memory storage in vivo. Here we investigated PKMζ stability in rat neurons to better understand its role during information encoding and storage.

TAK1-mediated autophagy and fatty acid oxidation prevent hepatosteatosis and tumorigenesis.

The MAP kinase kinase kinase TGFβ-activated kinase 1 (TAK1) is activated by TLRs, IL-1, TNF, and TGFβ and in turn activates IKK-NF-κB and JNK, which regulate cell survival, growth, tumorigenesis, and metabolism. TAK1 signaling also upregulates AMPK activity and autophagy. Here, we investigated TAK1-dependent regulation of autophagy, lipid metabolism, and tumorigenesis in the liver.

Improving signal to noise in labeled biological specimens using energy-filtered TEM of sections with a drift correction strategy and a direct detection device.

Energy filtered transmission electron microscopy techniques are regularly used to build elemental maps of spatially distributed nanoparticles in materials and biological specimens. When working with thick biological sections, electron energy loss spectroscopy techniques involving core-loss electrons often require exposures exceeding several minutes to provide sufficient signal to noise. Image quality with these long exposures is often compromised by specimen drift, which results in blurring and reduced resolution.

Fluorescent and photo-oxidizing TimeSTAMP tags track protein fates in light and electron microscopy.

Protein synthesis is highly regulated throughout nervous system development, plasticity and regeneration. However, tracking the distributions of specific new protein species has not been possible in living neurons or at the ultrastructural level. Previously we created TimeSTAMP epitope tags, drug-controlled tags for immunohistochemical detection of specific new proteins synthesized at defined times.

Isoform-specific targeting of PKA to multivesicular bodies.

Although RII protein kinase A (PKA) regulatory subunits are constitutively localized to discrete cellular compartments through binding to A-kinase-anchoring proteins (AKAPs), RI subunits are primarily diffuse in the cytoplasm. In this paper, we report a novel AKAP-dependent localization of RIα to distinct organelles, specifically, multivesicular bodies (MVBs). This localization depends on binding to AKAP11, which binds tightly to free RIα or RIα in complex with catalytic subunit (holoenzyme).

ChChd3, an inner mitochondrial membrane protein, is essential for maintaining crista integrity and mitochondrial function.

The mitochondrial inner membrane (IM) serves as the site for ATP production by hosting the oxidative phosphorylation complex machinery most notably on the crista membranes. Disruption of the crista structure has been implicated in a variety of cardiovascular and neurodegenerative diseases. Here, we characterize ChChd3, a previously identified PKA substrate of unknown function (Schauble, S.

Structure-function studies of blood and air capillaries in chicken lung using 3D electron microscopy.

Avian pulmonary capillaries differ from those of mammals in three important ways. The blood-gas barrier is much thinner, it is more uniform in thickness, and the capillaries are far more rigid when their transmural pressure is altered. The thinness of the barrier is surprising because it predisposes the capillaries to stress failure.

The C-terminus of connexin43 adopts different conformations in the Golgi and gap junction as detected with structure-specific antibodies.

The C-terminus of the most abundant and best-studied gap-junction protein, connexin43, contains multiple phosphorylation sites and protein-binding domains that are involved in regulation of connexin trafficking and channel gating. It is well-documented that SDS/PAGE of NRK (normal rat kidney) cell lysates reveals at least three connexin43-specific bands (P0, P1 and P2). P1 and P2 are phosphorylated on multiple, unidentified serine residues and are found primarily in gap-junction plaques.

Toxicity of Cr(lll) to Shewanella sp. strain MR-4 during Cr(VI) reduction.

Bioremediation of chromium through the reduction of hexavalent chromium (as the chromate ion, CrO42-) is based on the notion that the product, trivalent chromium (Cr(III)), is less toxic than chromate. In this study, we show that soluble Cr(III), present at pH 6-8 as the Cr3+ ion and/or hydroxyl complexes (henceforth referred to as uncomplexed Cr(III)), can be found transiently in significant concentrations and has a deleterious effect on Shewanella sp. MR-4.

Regional optic nerve damage in experimental mouse glaucoma.

Purpose: To assess the relationship between regional variation of axon loss and optic nerve head anatomy in laser-induced experimental glaucoma in the mouse.

Methods: Experimental glaucoma was induced unilaterally in eight NIH Swiss black mice. Intraocular pressure (IOP) was measured for 12 weeks, and the mice were killed.

Automated most-probable loss tomography of thick selectively stained biological specimens with quantitative measurement of resolution improvement.

We describe the technique and application of energy filtering, automated most-probable loss (MPL) tomography to intermediate voltage electron microscopy (IVEM). We show that for thick, selectively stained biological specimens, this method produces a dramatic increase in resolution of the projections and the computed volumes versus standard unfiltered transmission electron microscopy (TEM) methods. This improvement in resolution is attributed to the reduction of chromatic aberration, which results from the large percentage of inelastic electron-scattering events for thick specimens.

Optic nerve damage in mice with a targeted type I collagen mutation.

Purpose: Transgenic (Col1a1(r/r)) mice gradually develop elevated intraocular pressure (IOP) with open angles. The present study was undertaken to evaluate optic nerve axonal loss with time in these mice.

Methods: The IOP of transgenic (Col1a1(r/r)) mice and control wild-type (Col1a1(+/+)) mice was measured at 7, 12, 16, 24, 36, and 54 weeks of age using a microneedle method.

Optic nerve damage in experimental mouse ocular hypertension.

Purpose: To evaluate optic nerve damage in mice after laser-induced ocular hypertension.

Methods: Ocular hypertension was induced unilaterally in 13 NIH Black Swiss mice by laser photocoagulation of the limbus. Over the following 12 weeks, intraocular pressure (IOP) was measured at regular intervals by the microneedle method.

Cometabolism of Cr(VI) by Shewanella oneidensis MR-1 produces cell-associated reduced chromium and inhibits growth.

Microbial reduction is a promising strategy for chromium remediation, but the effects of competing electron acceptors are still poorly understood. We investigated chromate (Cr(VI)) reduction in batch cultures of Shewanella oneidensis MR-1 under aerobic and denitrifying conditions and in the absence of an additional electron acceptor. Growth and Cr(VI) removal patterns suggested a cometabolic reduction; in the absence of nitrate or oxygen, MR-1 reduced Cr(VI), but without any increase in viable cell counts and rates gradually decreased when cells were respiked.

Bid, Bax, and lipids cooperate to form supramolecular openings in the outer mitochondrial membrane.

Bcl-2 family proteins regulate the release of proteins like cytochrome c from mitochondria during apoptosis. We used cell-free systems and ultimately a vesicular reconstitution from defined molecules to show that outer membrane permeabilization by Bcl-2 family proteins requires neither the mitochondrial matrix, the inner membrane, nor other proteins. Bid, or its BH3-domain peptide, activated monomeric Bax to produce membrane openings that allowed the passage of very large (2 megadalton) dextran molecules, explaining the translocation of large mitochondrial proteins during apoptosis.