(12o) Molecular Engineering for Cellular Imaging: From Fluorescence to Magnetic Resonance

Research Interests: Light-emitting proteins such as luciferase and GFP (green fluorescent protein) have revolutionized biological research by enabling genetically targeted labeling and optical imaging of cells and biomolecules. However, rapid advances in synthetic biology have imposed increasingly challenging requirements for improved imaging technologies that can enable the study of cellular function within several distinct contexts relevant to disease, healthcare, and industrial bioproduction. Addressing these requirements necessitates the development of smart molecular reporters for multiple imaging modalities, providing an exciting opportunity for biomolecular engineering. My research aims to meet this challenge through two distinct approaches involving molecular engineering of: (1) reporter genes for magnetic resonance imaging (MRI) and (2) O₂-independent fluorescent reporters for optical imaging in low-O₂ conditions.

Research Experience: 

(1) Reporter genes for noninvasive cellular imaging using MRI (Advisor: Mikhail G. Shapiro, Caltech)

MRI is a widely established modality for noninvasive imaging of intact animals and patients. However, the lack of sensitive molecular reporters for MRI (analogous to GFP and luciferase for optics), represents a critical missing capability for extending MRI to the study of cellular functions in whole organisms. In this regard, we recently introduced diffusion altering reporter genes that render cells visible in diffusion-weighted magnetic resonance images simply by increasing the rate of water exchange across the cell membrane¹. This was achieved by overexpressing the human water channel, aquaporin, which results in robust MRI contrast without any deleterious effects on cell viability. Notably, aquaporin-based contrast is readily detectable at nanomolar levels of expression and in small (ó ??â??10%) subsets of aquaporin-labeled cells in mixed populations, which places aquaporin among the most sensitive reporter genes for MRI. Finally, we demonstrated the potential utility of aquaporin by imaging dynamic gene expression in a mouse brain tumor model.

(2) Engineering O₂-independent fluorescent reporter genes (Advisor: Charles M. Schroeder, UIUC)

The significance of low O₂ tensions (hypoxia & anoxia) is illustrated by the critical role of hypoxia in regulating bacterial pathogenesis, antibiotic sensitivity, gut microbiome dynamics, and cancer chemoresistance. Consequently, live cell imaging in low-O₂ environments is of tremendous interest in biomedical science. However, widely prevalent GFP and luciferase-based reporters show O₂-dependent light emission, which precludes robust quantitative cell imaging. On the other hand, alternative O₂-independent reporters based on flavin-binding photosensory proteins (FbFPs) are limited by poor brightness, stability, toxicity, a small library size, and an incomplete understanding of their performance and properties as cellular reporters². To this end, a key outcome of my research has been the characterization³, expansion, and diversification of the O₂-independent FbFP fluorescent palette using directed evolution⁴ and genome mining⁵. Notably, we engineered four new FbFP variants, which display improved brightness, photostability, small size (half the size of GFP), and a broad operational pH and thermal range (pH 3-11, up to 60°C).

Future Research Directions:

The transformative impact that optical reporters have had on biology can be attributed to the rich repertoire of cellular biosensors that have been developed based on foundational members of the GFP family. In contrast, there remains a critical dearth of genetically encoded MRI and optical sensors for cellular imaging in live animals and hypoxia respectively. As an independent investigator, I am interested in leveraging the previously described foundational classes of MRI and optical reporter genes to develop viable sensors for studying cellular function. Initial projects in the Mukherjee laboratory will have the following aims.

Aim 1: O₂-independent fluorescent biosensors. I propose to develop FbFP-based biosensors for imaging bacterial second messengers and cellular energy in physiologically realistic hypoxic models of bacterial infection, antibiotic resistance⁶, and host-pathogen interactions. Concurrently, we will explore new approaches for further enhancing FbFP performance through improvements in protein folding, solubility, chromophore binding, and enriching cellular flavin content.

Aim 2: Functional MRI reporters. I propose to expand the library of diffusion altering reporter genes through the development, characterization, and application of pH and phosphorylation-sensitive aquaporins for imaging pH fluctuations and cell signaling events in preclinical animal models of infection, disease, and injury. Furthermore, I am interested in repurposing a recently described eukaryotic Mn²⁺ binding protein to develop a genetically encoded MRI-based sensor for calcium imaging in vivo.

In summary, I envision the proposed research to culminate in the development of improved imaging tools as well as platform technologies that will enable new approaches to study cellular function and gene expression in the context of realistic in cellulo and in vivo models.

Teaching Interests: 

My teaching interests encompass topics in biomolecular engineering with a focus on imaging and therapy. Specifically, I am interested in developing and introducing a cross-disciplinary course on Molecular Engineering for Imaging and Therapy that integrates fundamental principles from chemical kinetics, thermodynamics, and imaging along with techniques in molecular biology and protein engineering with an overall emphasis on the development of molecular and cell-based sensors and therapeutics. A central goal of my teaching philosophy is to help students identify exciting new avenues for innovation and apply principles from biomolecular engineering to brainstorm creative solutions. Through this initiative, I seek to emphasize the importance of cross-disciplinary approaches in biomolecular engineering while encouraging students to identify and appreciate the foundational engineering and scientific principles that bioengineering is built upon.

Teaching Experience:

In my PhD, I worked as a teaching assistant for four semesters during which time I extensively lectured courses in Process Control, Biomolecular Engineering, and Bioenergy Technology for which I received the campus nomination for â??List of Teachers ranked Excellentâ?. During my postdoc, I was a guest lecturer for topics on MRI contrast agents and relaxation theory as part of a course in Molecular Imaging. In addition, I have mentored three undergraduate researchers of whom two are currently pursuing graduate studies in chemical and biomolecular engineering.

Publications:

1. Mukherjee, A., et al. Diffusion altering reporter genes for magnetic resonance imaging. In revision (preprint available on bioarXiv)

2. Mukherjee, A. et al. Flavin-based fluorescent proteins: Emerging Paradigms in Biological Imaging. Curr. Op. Biotech.. (2014)

3. Mukherjee, A., et al. Characterization of flavin-based fluorescent proteins: an emerging class of powerful fluorescent probes. PLOS ONE. (2013)

4. Mukherjee, A., et al. Directed evolution of bright mutants of a flavin-binding fluorescent protein from Pseudomonas putida. J. Biol. Eng. (2012).

5. Mukherjee, A., et al. Engineering and characterization of LOV-based fluorescent reporter proteins in C. reinhardtii & V. frigida. ACS Syn. Biol.(2014)

6. Mohan, R.*, Mukherjee, A.*, et al. A Multiplexed Microfluidic Platform for Antibiotic Susceptibility Screening. Biosens. Bioelectron. (2013) (* co-first)