Daniela Boassa, Ph.D. - US grants

DESCRIPTION (provided by applicant): We propose to refine and exploit powerful new genetically encoded labeling systems to visualize proteins by electron microscopy (EM), correlated with light microscopy (LM). EM is one of the most powerful techniques to see cell structures below optical resolution, but has suffered from lack of generally applicable genetically encoded labels until our recent development of miniSOG, a small flavoprotein that will do for EM what Green Fluorescent Protein did for LM. We aim to quantify the sensitivity and spatial resolution of miniSOG and to extend its applicability to low-temperature methods for sample preparation and imaging. Alternative genetically encoded labels will be developed and characterized to allow for two or more proteins of interest to be distinguished in a single EM image by electron energy loss spectroscopy of reaction deposits from oxidation of diaminobenzidine conjugated to different lanthanides. We have also developed reporters, based on the drug- controllable cis-acting protease from hepatitis C viral protease, to distinguish between old and newly synthesized copies of a genetically specified protein of interest. These fusion tags are visible by correlated LM and EM and will be applied to plasticity and disease-related synaptic proteins to reveal their localized appearance and turnover, initially in culture bt eventually in intact mammalian brain. Viral and Cre/lox modular targeting vectors will be created to make cell-type-selective expression of the above EM/LM tags robust and widely applicable to systems as complex as mouse models of disease and learning. We have chosen cell types and proteins important in liver fibrosis and synaptic plasticity as test cases because these biological processes are diverse, engage outstanding local collaborators, and have great biomedical importance. With collaborators we will investigate how hepatocytes, hepatic stellate cells, and endothelial cells change ultrastructural morphology and location of key proteins during fibrogenic injury. Another collaboration will focus on activity-induced changes in morphology and adhesion molecules at synapses in the amygdala during fear conditioning and memory consolidation. Such extension of these new tools into transgenic animals will make it possible to relate ultrastructural location and metabolic turnover of genetically specified proteins to whole- animal behavior and disease.

PROJECT SUMMARY We propose to refine and exploit powerful new markers and labeling systems to visualize multiple proteins or other biomolecules by electron microscopy (EM), correlated with light microscopy (LM). EM is one of the most powerful techniques to see cell structures below optical resolution, but has suffered from lack of generally applicable genetically encoded labels until our recent development of new EM-compatible markers such as miniSOG, a small flavoprotein that will do for EM what Green Fluorescent Protein did for LM, and APEX, an engineered ascorbate peroxidase. We have developed split-miniSOG, two fragments that do nothing separately, but when brought back together reversibly regenerate miniSOG and its fluorescence and photooxidative capability. Our newly developed split-miniSOG complementation system allows us to visualize intermolecular interactions at high resolution by EM. We have developed new strategies and techniques to improve the acquisition of element-specific analytical maps in transmission EM to achieve what we refer to as multicolor EM. This technology allows distinct EM- level labeling of multiple species with a different lanthanide element that is separately imaged by electron energy-loss spectrometry (EELS) and displayed in a distinct pseudocolor. Just as multicolor fluorescence has been vital to understanding many cellular functions at optical resolution, we anticipate multicolor EM will be valuable at finer resolution. Our overall goals are to expand and improve EM-compatible reporters, `molecular painting' chemistry, and new instrumentation to improve the resolution, sensitivity, and specificity with which multiple proteins or other biomolecules can be imaged by EM. We aim to obtain a genetically encoded far-red or near-infrared diaminobenzidine (DAB) photooxidizer analogous to miniSOG but excited at significantly longer wavelengths to facilitate multispecies labeling at high resolution. We will develop controlled living polymerization as an alternative to photooxidative amplification using modern methods such as ATRP, ROMP, or RAFT applied to fixed cells and tissue, to generate lanthanide-containing polymers of defined length and morphology at desired cellular targets. As test cases, these techniques will be applied to fundamental biological problems such as the spatial organization of the genome in the nucleus and aggregation of specified proteins involved in neurodegenerative diseases. We have chosen these biological processes because they are diverse, engage outstanding collaborators, and have great biomedical importance. Ultimately, the combination of photooxidizing, peroxidase-based, and nonphotochemical amplifying systems will give cell and molecular biologists a rich palette for EM, comparable to small-molecule fluorophores plus fluorescent proteins for optical microscopy.

PROJECT SUMMARY Synaptic dysfunction is a common feature of neuropsychiatric disease. For example, a hallmark of age-related neurodegenerative diseases such as Alzheimer?s and Parkinson?s is synaptic fibrilization and aggregation of key proteins that participate in synapse and cell loss. Maladaptive plastic changes in synapse structure and function underlie key aspects of behavioral and mood disorders ranging from addiction to depression, as well as neurodevelopmental diseases like schizophrenia and autism. It is for these reasons that many investigators across a range of neuroscience disciplines study the synapse, and the reason that new tools to study synapse structure and function within neural circuits of interest are sorely needed. Indeed, current tools to assess synapse structure in defined cell types are not readily compatible with state-of-the-art 3D volume approaches such as serial block face scanning electron microscopy, and are severely hampered by inadequate computational tools for quantitative assessment of these massive datasets. However, advances in molecular genetics, optics, engineering and computing provide new opportunities to develop information rich strategies to peer into the synapse. Here, we combine such advances to achieve a new state-of-the-art in imaging and analyzing microcircuit connectivity and synapse structure within neurotransmitter-defined neural networks. Specifically, we leverage the fact that the bulk of signaling across the synapse is mediated by a relatively small population of small molecule neurotransmitters that are synthesized and packaged into synaptic vesicles at the site of release in axonal compartments. The bulk of neurotransmission is thus dependent on just seven well- described vesicular transporters expressed in brain. Our overall goal is to build a rigorous, easily deployable, cell-type-specific, expandable, multi-functional toolkit for imaging and quantifying neurotransmitter-defined synaptic connections by both light and electron microscopy in mice. To accomplish this, we will use CRISPR/Cas9 to insert electron microscopy-compatible tags into native vesicular transporters (Aim 1), establish simplified procedures for their monochrome and ?multicolor? labeling in 3D ultrastructure (Aim 2), and computational tools for automated segmentation and quantitative analysis of key pre- and post-synaptic metrics (Aim 3). Though these Aims are independently meritorious, by synthesizing them we aim to generate a complete toolkit that will allow investigators to render neurotransmitter-defined circuit connections into 3D ultrastructure datasets with automated quantitative assessment of key features of pre- and post-synaptic structure.

Project Summary/Abstract Chlamydia genital infections are the most commonly reported infection in the U.S. All Chlamydia species cause an unusual intracellular infection in which there is conversion between a dividing form of the bacterium (reticulate body or RB) and the infectious form (elementary body or EB). RB-to-EB conversion is critical for producing infectious progeny that can spread the infection to a new host cell, but the mechanisms that regulate it are unknown. There has been a longstanding assumption in the Chlamydia field that conversion is regulated by an extrinsic factor. However, we have obtained data to support a new regulatory mechanism in which RB size is used as an intrinsic factor to control conversion. Based on temporal measurements of chlamydial size and number obtained with three-dimensional electron microscopy (3D EM), we hypothesize that RBs undergo size reduction through successive rounds of replication and can only convert into an EB below a size threshold. In Aim 1, we will test this size control mechanism by determining whether RB size is altered when the timing of RB-to-EB conversion onset is changed. In Aim 2, we will investigate if the size of the first RB plays a role in starting the timer of RB size reduction that eventually culminates in RB-to-EB conversion. In Aim 3, we will study how RB size could be used to regulate conversion. We propose a titration mechanism in which EUO, a repressor of late chlamydial genes, is titrated away in smaller RBs to promote conversion. In Aim 4, we will study alternative mechanisms that utilize extrinsic signals to control conversion. Using mathematical modeling and 3D EM analysis, we will test a contact-dependent mechanism, which is based on contact of the RB with the inclusion membrane as the external signal, and a chlamydial communication mechanism in which the external signal is produced by other chlamydiae. These studies will provide important information about the mechanism of RB-to-EB conversion that can be applied in new therapeutic strategies to block the developmental cycle and the production of infectious progeny.