Single-Cell Molecular Pathway Analysis in Aging Systems via Novel Mass Cytometry Methods

Different cell types, each with unique composition, properties, and functions, are essential to sustain life in an organism. Bulk analysis of such heterogeneous mixtures of cells does not distinguish the characteristics of a given cell type or differences between cells within the same type, missing information that is key to understanding molecular mechanisms foundational to the rational design of therapies for disease and ailments. The goal of this proposal is to develop single-cell and imaging methods using metal-bearing probes to monitor metabolic processes in live cells such as siRNA delivery, protein synthesis, and post-translational modification. The cells will then be fixed and processed for subsequent detection of protein and mRNA markers. There are three aims: (1) Delivering molecular probes to viable cells to explore molecular pathways in single cells by CyTOF. (2) Barcoding prenylation probes to explore molecular pathways in mixed samples of single cells. (3) Exploring the effect of aging in murine primary cells by CyTOF and murine tissue by MIBI-ToF. The proposed work will expand the use of multi- parametric capabilities of mass cytometry (i.e. cytometry by time-of-flight, CyTOF, and multiplexed ion beam imaging by time-of-flight, MIBI-ToF) in the biotechnological and biomedical fields. The development of these methodologies will explore the causal relationship between autophagy and senescence in murine liver models, exploring the hypothesis that the mevalonate pathway provides a link between the two. The ultimate goal is to understand the relationship between autophagy, mevalonate, and senescence pathways in aging and inform emerging treatments to counteract the detrimental effects of senescent cells.

Novel Approaches to Investigate Autophagy

Autophagy is a vital cellular process responsible for the degradation of subcellular components including organelles. We aim to define alterations in autophagy pathways associated with aging and disease. To pursue this aim, we are developing bioanalytical technologies, such as individual-organelle capillary electrophoresis, and develop new strategies based on ‘omic’ platforms including quantitative proteomics, metabolomics, and mass cytometry. In our proteomic strategies we put emphasis on the characterization of post-translational modifications such ubiquitination and prenylation, and on single-cell proteomic analysis, which is done by mass cytometry.

Bioanalytical Microfluidic / Nanoporous Devices for Biological Matters 

Heterogeneity in organelle traits  has been associated with devastating human maladies such as neurodegenerative diseases or cancer. Therefore, assessing subpopulation of organelles is imperative to understand the biomolecular foundations of these diseases. We develop miniaturized bioanalytical devices that can be potential tools fostering biomedical studies. Associated subjects including biomolecular labeling i.e., SELEX, microfluidic technologies, or pathogen sensing are studied. 

Multi-Dimensional Analysis of Individual Organelles

Traditional methods for the analysis of complex intracellular process are bulk measurements which mask organelle heterogeneity and complicate the analysis of inter-organelle association and trafficking. Thus, methods for individual organelle quantification are needed to address these deficiencies. Current techniques for quantifying individual autophagy organelles are either low through-put or are dimensionally limited. We make use of the multiparametric capability of mass cytometry to investigate phenotypic heterogeneity. We also focus on development of clustering algorithm for high-dimensional data and computational approaches to rarer cell identification, enabling advanced single-cell analysis. 

Mitochondrial Properties and Heterogeneity in Age- and Disease-Related Systems

Mitochondria are highly dynamic and heterogeneous organelles, which may be classified into several subpopulations. How such subpopulations differ in composition and biochemical function is mostly unknown. It has been proposed that only some of such subpopulations are associated with mitochondrial dysfunction. We aim to define the properties of such subpopulations through their isolation and subsequent analysis. Relevant methods, currently under development, include those for characterization of mitochondrial DNA damage and aptamers for affinity enrichment of mitochondria.

Analysis of Redox, Reactive Oxygen Species and Oxidative Damage to Biological Systems

Despite the logical associations between redox status, the production of reactive oxygen species (ROS), and the appearance of oxidative damage in a subcellular dependent manner, there is great need to provide quantitative assessments and measurements of these three aspects of cellular and subcellular biology. These advancements are critical to fundamental aspects of ROS-based signaling and regulation of oxidative damage. Our goal is to design novel bioanalytical strategies to quantify redox status, different types of ROS, and oxidative damage in multiple models of aging and disease. We are currently involved in the development of novel redox probes and are using micellar-electrokinetic chromatography (MEKC) to measure multiple types of ROS species in the same analytical separation.

Electrokinetic Behavior of Biological Particles

Our research team introduced the analysis of individual organelles by capillary electrophoresis. This is a powerful strategy to represent distributions of electrophoretic mobility and isoelectric point of isolated individual organelles. Because these organelles are membrane-bound biological particles, these strategies can be extended to other particles of similar nature. Provided control of the separation conditions, it is also feasible to investigate the electrokinetic behavior of such particles.