Melike Lakadamyali, Ph.D. - US grants

Project Summary In this proposal, we will develop new methods to overcome one of the main challenges in super-resolution microscopy and enable quantification of protein copy number at the nanoscale level. Super-resolution microscopy is an enabling tool that reveals the subcellular organization of molecular complexes with unprecedented spatial resolution. The nanoscopic organization of these complexes into functional units is highly important for regulating their subcellular activity in a spatially and temporally controlled manner. However, it has been very challenging to quantify the protein copy number composition of multi-protein complexes. Protein copy number is highly important for regulating or mis-regulating protein function. Proteins may be functional below a certain oligomeric assembly and gain toxic function when their oligomeric composition crosses a critical threshold leading to diseases. Therefore, the ability to properly quantify the sub- cellular copy number distribution of proteins within molecular complexes is important for gaining mechanistic insight into healthy and diseased function of these proteins. The main challenge, in doing so, is to overcome the artefacts arising from the unknown labeling stoichiometry and complex fluorophore photophysics. We have made important leaps towards overcoming this challenge by developing calibration nanotemplates for super-resolution microscopy. However, the lack of standard, easy-to-use methods and reagents that account for variability in experimental conditions has made it difficult for non-experts to adapt these developments. Therefore, there is an immediate need for highly standardized methodologies that allow protein copy number quantification independent of experimental conditions. The goal of this proposal is to address this big challenge and establish a versatile, easy-to-use and universal calibration method that can be adapted by the scientific community to quantify the copy number distribution of any protein of interest. Our aims are: (i) to develop calibration nanotemplates for both small and large protein complexes using DNA origami as well as novel nanotemplates that use designer protein nanocages, (ii) to acquire calibration data for diverse, super-resolution compatible labeling strategies and identify calibration functions, (iii) to develop the innovative concept of standardization based on novel use of benchmarking standards and methods that can transform the calibration functions among different experimental conditions and (iv) to develop an all-integrated, user-friendly, open- source, modular software that incorporates all the steps from single molecule localization to protein copy number determination. This proposal has the potential to advance super-resolution microscopy from a mainly descriptive tool into the era of quantitative and mechanistic cell biology. As one specific example, the methods developed here will make it possible to reveal the sub-cellular distribution and evolution of protein aggregation in a highly quantitative manner in several disease states at much earlier time points than has been possible thus far, potentially enabling new diagnostic and drug screening methods in the future. We expect the method to be widely applicable to a large number of biomedical questions and have a broad impact.

Numerous organisms from yeast to humans organize their genome by wrapping it repeatedly around histone proteins into nanoscale spools known as chromatin. Cells use the organization of chromatin to dictate whether a gene is actively expressed or turned off. Combining the ability to target a specific gene, visualize its location and structure, activate the gene, and detect gene expression in live cells would be a major technological advance in how genes are studied and controlled in living organisms, and lead to applications in many fields, including medicine, agriculture, energy and the environment. DNA nanotechnology, which uses well-understood folding properties of DNA to engineer nanoscale, biocompatible structures, is an emerging technology with the potential to combine these functions. A 5-PI team will apply bioengineering, cell biology, genetics, single molecule spectroscopy, super resolution microscopy and multi-scale molecular modeling to develop such DNA-based nanodevices that can also operate in live cells and be "switchable" to allow these functions to be triggered at will. The research will be integrated into university curricula, and will enable cross-disciplinary, collaborative and international training of graduate students. The PIs will also broaden participation of underrepresented students in STEM by creating open access standards-based videos and modules for use by K-12 teachers.

Recent advances in genetic and epigenetic methods have enabled chromatin engineering technologies that 1) target genes to 2) visualize chromatin structure, 3) activate target genes, and 4) detect gene-specific transcription. However, current tools (including super-resolution imaging, chromatin conformation capture and genome engineering with CRISPR/Cas9) are usually only able to accomplish one of these functions at a time, giving single-channel, static views of the players and processes at a specific transcription site. This project will leverage DNA nanotechnology to leapfrog current technologies for probing and engineering genome and epigenome functions. A team of five PIs will develop multi-functional DNA origami (DO) nanodevices that combine targeting, functional modifications and RNA detection onto a single platform, operate in live cell nuclei, and are "switchable", allowing for real-time detection and for functions to be triggered by endogenous or external signals. The outcomes will serve as a foundation for future automated devices that target and regulate genome and epigenome functions, including gene expression, and serve as diverse new toolsets for science, engineering, and medical applications.

This award is co-funded by the Genetic Mechanisms cluster in the Division of Molecular and Cellular Biosciences in the Biological Sciences Directorate, the Emerging Frontiers in Research and Innovation program in the Division of Emerging Frontiers and Multidisciplinary Activities in the Engineering Directorate, and the Chemistry of Life Processes program in the Division of Chemistry in the Mathematics and Physical Sciences Directorate.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

PROJECT SUMMARY This RM1 proposal focuses on defining and characterizing the integrated systems that support intracellular organelle transport and placement. Rather than just characterizing ?molecules? as biophysicists or ?organelle dynamics? as cell biologists, our goal is to achieve an atomic-to- cellular-to-in vivo level understanding of the mechanisms of organelle transport. Our collaborative and multi-disciplinary research team will discover and study the native microenvironments in cells and in vitro, by considering physiologically-relevant combinations of molecular motors, filaments, adaptors, membranes and other cofactors that control organelle movement and polarization. We will use mitochondria as our initial model system, as many key molecules and signaling pathways that are essential for the dynamics of this organelle have already been described. We will uncover the roles of spatial organization and dynamic assembly of filaments, mechanical forces, motor activity, and other regulatory elements will be investigated. We will reconstitute complex microenvironments in vitro to determine motile and anchoring mechanisms. We will computationally model these systems and experimentally test models directly in cells and in vivo. Our research team will be energized and expanded by implementing an Exploratory Pilot Studies Program to incorporate promising early-stage- investigators into our collaborative team.

Short tandem repeat regions (STR) are distributed evenly across the human genome, and recent genome-wide studies have demonstrated that STRs are polymorphic across individuals and linked to gene expression levels. STR instability at key genomic loci has been causally linked to disease pathophysiology in a range of expansion disorders. We recently demonstrated that nearly all disease-associated STRs co-localize with boundaries demarcating topologically associated domains (TADs). Moreover, we have observed that pathologic STR instability and transcriptional silencing can destroy the associated boundary and shift genomic loci to the nuclear periphery. These results now open critical unanswered questions regarding whether and how STR expansion and pathologic alterations in gene expression are functionally linked to boundary integrity and radial positioning. Here, we focus on the prototypic repeat expansion disorder Friedreich?s ataxia (FRDA) in which expansion of a GAA STR in the first intron of the FRATAXIN (FXN) gene results in cardiac and neuronal pathology. The cardiac pathology, specifically hypertrophy, fibrosis, and occasional dilation of the ventricle, is the etiology of significant FRDA mortality. GAA expansion is associated with the silencing of FXN transcription and a repositioning of the locus to the nuclear periphery. However, it remains unclear if the change in genome folding, radial positioning, or reduced expression drives STR expansion or vice versa. A major technical barrier contributing to this knowledge gap is that STR instability and genome folding are classically evaluated in bulk populations, however they exhibit tremendous variation across individual somatic cells of the same subtype and among cell types within a pathologically affected tissue. Here, we seek to decipher the causal link among STR instability, transcription, radial positioning, and genome folding. Our central hypothesis is that disruption of long-range loops is the initial event triggered by STR expansion leading to a cascade of heterochromatin spreading, silencing, and loss of radial positioning. We will test our hypothesis by generating genome-wide, single-cell maps of chromatin accessibility, expression, and the repressive H3K9me3 heterochromatin mark in GAA-expanded and control iPS cells and iPS-derived cardiomyocytes. We will integrate genomics data with single-cell sequential Oligopaints/OligoSTORM imaging of TADs and local chromatin structure, as well as single molecule RNA FISH for FXN expression. We will implement multiple genome engineering strategies, including dCas9-VP64 FXN activation and dCas9-CTCF loop re-engineering in FRDA GAA-iPS cells, and dCas9-Krab-Dnmt3a FXN silencing and dCas9-Krab CTCF-mediated loop disruption in healthy iPS cells. We will assay the effect of genome engineering approaches on TADs, radial positioning, STR length, and FXN expression in single cells. Successful completion of the proposed work will shed light on the pathophysiological mechanisms underlying repeat expansion disorders by deciphering the cause-and-effect relationships among genome folding, radial positioning, transcription, and STR expansion.