From Single-Cell to Whole-Body: Developing a Molecular Neuroscience Toolkit

Throughout my Ph.D. I have worked on technology development, at first to answer basic scientific questions and eventually for therapeutic applications. This technology development applied to a variety of fields, from neuroscience to development to gene therapy, and acted upon biological systems in a wide range of scale, from the single-cell monitoring to organism-wide gene-transfer. My graduate research began with the engineering of microbial rhodopsin spectral properties and fluorescence. By making use of their ability to absorb light and emit fluorescence in a voltage-dependent manner, I aimed to interrogate neuronal activity during behavior at the single-cell level. That line of research ended with publication of the voltage-sensor Archer, which I used to track activity of a single cell in vivo in awake, behaving worms. I then shifted from tracking activity at the single cell level, to visualizing entire organisms, by developing clearing techniques that enable a high-resolution, three-dimensional analysis of a diverse range of tissues. I began by optimizing tissue-clearing parameters for various tissue types and a wide variety of experimental needs. I then took that knowledge and applied it to visualizing and tracking the developing neural crest in cleared, whole-mount chicken embryos, discovering some unexpected derivates. Finally, I became interested not only in visualizing entire organisms, but in developing technologies to facilitate gene transfer throughout the body. The rapidly growing field of gene therapy is in constant need of new tools that target specific tissues, avoiding off-target effects. The end of my Ph.D. has been spent engineering viruses that can be delivered body-wide, but target only specific areas of therapeutic interest, like the brain and lungs.</p

Broad gene expression throughout the mouse and marmoset brain after intravenous delivery of engineered AAV capsids

Genetic intervention is increasingly explored as a therapeutic option for debilitating disorders of the central nervous system. The safety and efficacy of gene therapies relies upon expressing a transgene in affected cells while minimizing off-target expression. To achieve organ/cell-type specific targeting after intravenous delivery of viral vectors, we employed a Cre-transgenic-based screening platform for fast and efficient capsid selection, paired with sequential engineering of multiple surface-exposed loops. We identified capsid variants that are enriched in the brain and detargeted from the liver in mice. The improved enrichment in the brain extends to non-human primates, enabling robust, non-invasive gene delivery to the marmoset brain following IV administration. Importantly, the capsids identified display non-overlapping cell-type tropisms within the brain, with one exhibiting high specificity to neurons. The ability to cross the blood–brain barrier with cell-type specificity in rodents and non-human primates enables new avenues for basic research and potential therapeutic interventions unattainable with naturally occurring serotypes

Broad gene expression throughout the mouse and marmoset brain after intravenous delivery of engineered AAV capsids

Genetic intervention is increasingly explored as a therapeutic option for debilitating disorders of the central nervous system. The safety and efficacy of gene therapies relies upon expressing a transgene in affected cells while minimizing off-target expression. To achieve organ/cell-type specific targeting after intravenous delivery of viral vectors, we employed a Cre-transgenic-based screening platform for fast and efficient capsid selection, paired with sequential engineering of multiple surface-exposed loops. We identified capsid variants that are enriched in the brain and detargeted from the liver in mice. The improved enrichment in the brain extends to non-human primates, enabling robust, non-invasive gene delivery to the marmoset brain following IV administration. Importantly, the capsids identified display non-overlapping cell-type tropisms within the brain, with one exhibiting high specificity to neurons. The ability to cross the blood–brain barrier with cell-type specificity in rodents and non-human primates enables new avenues for basic research and potential therapeutic interventions unattainable with naturally occurring serotypes

Directed Evolution of Gloeobacter violaceus Rhodopsin Spectral Properties

Proton-pumping rhodopsins (PPRs) are photoactive retinal-binding proteins that transport ions across biological membranes in response to light. These proteins are interesting for light-harvesting applications in bioenergy production, in optogenetics applications in neuroscience, and as fluorescent sensors of membrane potential. Little is known, however, about how the protein sequence determines the considerable variation in spectral properties of PPRs from different biological niches or how to engineer these properties in a given PPR. Here we report a comprehensive study of amino acid substitutions in the retinal binding pocket of Gloeobacter violacaeus rhodopsin (GR) that tune its spectral properties. Directed evolution generated 70 GR variants with absorption maxima shifted by up to +/- 80 nm, extending the protein’s light absorption significantly beyond the range of known natural PPRs. While proton pumping activity was disrupted in many of the spectrally shifted variants, we identified single tuning mutations that incurrred blue and red shifts of 42 nm and 22 nm, respectively, that did not disrupt proton pumping. Blue-shifting mutations were distributed evenly along the retinal molecule while red-shifting mutations were clustered near the residue K257, which forms a covalent bond with retinal through a Schiff base linkage. Thirty-four of the identified tuning mutations are not found in known microbial rhodopsins. We discovered a subset of red-shifted GRs that exhibit high levels of fluorescence relative to the wild-type protein

Archaerhodopsin variants with enhanced voltage-sensitive fluorescence in mammalian and Caenorhabditis elegans neurons

Probing the neural circuit dynamics underlying behaviour would benefit greatly from improved genetically encoded voltage indicators. The proton pump ​Archaerhodopsin-3 (​Arch), an optogenetic tool commonly used for neuronal inhibition, has been shown to emit voltage-sensitive fluorescence. Here we report two ​Arch variants with enhanced radiance (Archers) that in response to 655 nm light have 3–5 times increased fluorescence and 55–99 times reduced photocurrents compared with ​Arch WT. The most fluorescent variant, Archer1, has 25–40% fluorescence change in response to action potentials while using 9 times lower light intensity compared with other ​Arch-based voltage sensors. Archer1 is capable of wavelength-specific functionality as a voltage sensor under red light and as an inhibitory actuator under green light. As a proof-of-concept for the application of ​Arch-based sensors in vivo, we show fluorescence voltage sensing in behaving Caenorhabditis elegans. Archer1’s characteristics contribute to the goal of all-optical detection and modulation of activity in neuronal networks in vivo

Directed evolution of a far-red fluorescent rhodopsin

Microbial rhodopsins are a diverse group of photoactive transmembrane proteins found in all three domains of life. A member of this protein family, Archaerhodopsin-3 (Arch) of halobacterium Halorubrum sodomense, was recently shown to function as a fluorescent indicator of membrane potential when expressed in mammalian neurons. Arch fluorescence, however, is very dim and is not optimal for applications in live-cell imaging. We used directed evolution to identify mutations that dramatically improve the absolute brightness of Arch, as confirmed biochemically and with live-cell imaging (in Escherichia coli and human embryonic kidney 293 cells). In some fluorescent Arch variants, the pK_a of the protonated Schiff-base linkage to retinal is near neutral pH, a useful feature for voltage-sensing applications. These bright Arch variants enable labeling of biological membranes in the far-red/infrared and exhibit the furthest red-shifted fluorescence emission thus far reported for a fluorescent protein (maximal excitation/emission at ∼620 nm/730 nm)

An EMT-Driven Alternative Splicing Program Occurs in Human Breast Cancer and Modulates Cellular Phenotype

Epithelial-mesenchymal transition (EMT), a mechanism important for embryonic development, plays a critical role during malignant transformation. While much is known about transcriptional regulation of EMT, alternative splicing of several genes has also been correlated with EMT progression, but the extent of splicing changes and their contributions to the morphological conversion accompanying EMT have not been investigated comprehensively. Using an established cell culture model and RNA–Seq analyses, we determined an alternative splicing signature for EMT. Genes encoding key drivers of EMT–dependent changes in cell phenotype, such as actin cytoskeleton remodeling, regulation of cell–cell junction formation, and regulation of cell migration, were enriched among EMT–associated alternatively splicing events. Our analysis suggested that most EMT–associated alternative splicing events are regulated by one or more members of the RBFOX, MBNL, CELF, hnRNP, or ESRP classes of splicing factors. The EMT alternative splicing signature was confirmed in human breast cancer cell lines, which could be classified into basal and luminal subtypes based exclusively on their EMT–associated splicing pattern. Expression of EMT–associated alternative mRNA transcripts was also observed in primary breast cancer samples, indicating that EMT–dependent splicing changes occur commonly in human tumors. The functional significance of EMT–associated alternative splicing was tested by expression of the epithelial-specific splicing factor ESRP1 or by depletion of RBFOX2 in mesenchymal cells, both of which elicited significant changes in cell morphology and motility towards an epithelial phenotype, suggesting that splicing regulation alone can drive critical aspects of EMT–associated phenotypic changes. The molecular description obtained here may aid in the development of new diagnostic and prognostic markers for analysis of breast cancer progression.National Institutes of Health (U.S.) (R01-HG002439)National Science Foundation (U.S.) (equipment grant)National Institutes of Health (U.S.) (Integrative Cancer Biology Program Grant U54-CA112967)David H. Koch Institute for Integrative Cancer Research at MIT (Ludwig Center for Metastasis Research)David H. Koch Institute for Integrative Cancer Research at MITMassachusetts Institute of Technology (Croucher Scholarship)Massachusetts Institute of Technology (Ludwig Fund postdoctoral fellowship)National Institutes of Health (U.S.) (NIH CA100324)National Institutes of Health (U.S.) (AECC9526-5267

Whole-body tissue stabilization and selective extractions via tissue-hydrogel hybrids for high-resolution intact circuit mapping and phenotyping

To facilitate fine-scale phenotyping of whole specimens, we describe here a set of tissue fixation-embedding, detergent-clearing and staining protocols that can be used to transform excised organs and whole organisms into optically transparent samples within 1–2 weeks without compromising their cellular architecture or endogenous fluorescence. PACT (passive CLARITY technique) and PARS (perfusion-assisted agent release in situ) use tissue-hydrogel hybrids to stabilize tissue biomolecules during selective lipid extraction, resulting in enhanced clearing efficiency and sample integrity. Furthermore, the macromolecule permeability of PACT- and PARS-processed tissue hybrids supports the diffusion of immunolabels throughout intact tissue, whereas RIMS (refractive index matching solution) grants high-resolution imaging at depth by further reducing light scattering in cleared and uncleared samples alike. These methods are adaptable to difficult-to-image tissues, such as bone (PACT-deCAL), and to magnified single-cell visualization (ePACT). Together, these protocols and solutions enable phenotyping of subcellular components and tracing cellular connectivity in intact biological networks

Fluorescent boost for voltage sensors

The development of a voltage sensor in which a microbial rhodopsin protein is fused with a fluorescent protein enables the neuronal activity of single cells in live animals to be measured with unprecedented speed and accuracy