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  • Stem cell | Cell Maturation | Microscopy

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  • Physician scientist | Technique development | Computational Biologist

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  • Cell cycle expertise | Resilient | Creative

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Uncovering mechanisms of exocrine cellular maturation

Jason Mills (mentor)

Washington University in St. Louis

Division of Gastroenterology, Department of Medicine

Undergraduate research

Submitted cover art for his research work related to MIST1 (PMC5322730)

Submitted cover art for his research work related to MIST1 (PMC5322730)

Stem cell background

Hei-Yong was always passionate about research. Prior to pursuing his graduate degree, he sought out numerous research opportunities. The bulk of his work was rooted in stem cell biology and cellular differentiation. He spent various summers in labs working on projects related to cancer stem cells eradication, induced pluripotent stem cell (iPSC) differentiation, and spermatagonial stem cell identification (Sachs et al. 2014).

Figure depicting the effects of MIST1 on cellular architecture of either zymogenic or parietal cells (PMC5322730).

Cell development and maturation

His passion brought him to the tutelage of Dr. Jason Mills at Washington University in St. Louis, where he worked as a research technician. There, he aided in the research of Paligenosis, a cell state where cells de-differentiate and re-enter a proliferative cell state as a result of cellular injury (Willet et al. 2018).

His primary undergraduate work uncovered a key mechanism of cellular maturation in exocrine cells regulated by transcription factor MIST1 (Lo et al. 2017). The loss of MIST1 resulted in collapsed cellular architecture in numerous exocrine cell types.

3D-reconstruction of cells in the stomach unit taken from a ~35 µm cube depicting wild-type mouse parietal cells (purple), mucous neck cells (yellow), chief cells (brown), capillaries (red), and nuclei (blue). PMC5322730

Microscopy enthusiast

Like MIST1 does for exocrine cells, Hei-Yong credits Dr. Mills and the experience working in the lab for allowing him to mature as a scientist. He developed numerous skills in his tenure, including organoid cell culture, mice dissection / tissue processing, and animal husbandry.

Most notably, he became an expert in microscopy techniques, from histological tissue preparation to staining and imaging. These skills precipitated in co-authored manuscripts (Moore et al. 2016; Jacome-Sosa et al. 2021).

His microscopy work culminated with the imaging of the first mouse stomach by focused ion beam scanning electron microscopy (FIB-SEM) (depicted in the video).

Developing tools to probe subcellular RNA

J. Matthew Taliaferro (mentor)

University of Colorado, Anschutz Medical Campus

Molecular Biology Program

Department of Biochemistry and Molecular Genetics

Work funded by MSTP T32, MOLB T32, the RNA Bioscience Initiative, and a W.M. Keck foundation grant.

Submitted and accepted cover art for his research work related to Halo-Seq (PMC9097300).

Physician Scientist training

Hei-Yong Lo sought to refine his research skills. He joined the Medical Scientist Training Program at the University of Colorado, Anschutz Medical Campus in pursuit of combining biomedical research with clinical outcomes. He believed that developing a mechanistic understanding of the molecular determinants of health and disease was critical for translational research.

This philosophy led him to pursue his PhD in Molecular Biology under the tutelage of J. Matthew Taliaferro (Taliaferrolab).

Workflow for Halo-seq (PMC9097300). Created with BioRender.

Technique development

As a graduate student, he studied basic cell biology, investigating the localization of RNAs within a cell (Engel et al. 2020). His worked aimed to uncover the identity of RNAs around different compartments within a cell, and the mechanistic determinants of how these RNAs localized to those subcompartments.

His work helped to develop a proximity labeling, Halo-seq (Engel et al. 2021; Lo et al. 2022). Halo-seq enriches for these RNAs with streptavidin beads, allowing for subsequent RNA sequencing.

Halo-seq has successfully been used to identify the subcellular RNAs in the nucleus, cytoplasm, nucleolus, and apical/basal spaces of enterocytes (Engel et al. 2021, Goering et al. 2023).

Always willing to improve

The efficacy of Halo-seq is limited by the use of streptavidin beads.

To improve upon Halo-seq, he further developed OINC-seq (Oxidation-Induced Nucleotide Conversion sequencing). OINC-seq directly detects oxidized lesions on RNAs with mutations created by reverse transcriptase (Lo and Goering et al, 2024, in revision). It does so without the need for subsequent biotinylation steps.

OINC-seq has been successful in identifying RNA in the nucleus, cytoplasm, inner/outer mitochondrial spaces, and endoplasmic reticulum.

It is also the first proximity labeling technique to date to label RNAs in real time in live zebrafish.

Coding competent

Processing large RNA-seq datasets requires an intimate understanding of sequencing combined with computational bioinformatic approaches. Hei-Yong uses a combination of Linux, Python, and RStudio to analyze bulk RNA-sequencing.

In addition to his primary projects, his bioinformatics expertise led him to contribute to numerous other projects. These projects including the validation of another proximity labeling technique (FAP-seq) (Li et al, 2024). He also helped to identify how polyadenylation changes RNA function (Espinosa et al. 2022).

He also was a teaching assistant for the biostatistics course for Molecular Biology, which utilizes all three programming languages.

Understanding RNA localization to the mammalian centrosome

J. Matthew Taliaferro (mentor)

University of Colorado, Anschutz Medical Campus

Molecular Biology Program

Department of Biochemistry and Molecular Genetics

Work funded by MSTP T32, MOLB T32, the RNA Bioscience Initiative, and a W.M. Keck foundation grant.

Centrosesome. Artwork by Hei-Yong Lo, 2024. 20 centrosomes superimposed on one another. 10 of those centrosomes are also superimposed in the center. Created with affinity designer. 

Cell cycle expertise

Returning to his stem cell roots, he turned his expertise towards identifying how localized RNAs impact cell cycle progression.

For decades, the identity of RNAs localized to the centrosome have eluded researchers. Identifying the local RNA content might uncover why these RNAs localize and how they do so.

Using both Halo-seq and OINC-seq, he sought to identify RNAs around a miniscule structure critical for mitosis – the centrosome. Unfortunately, his work joins the numerous other failed attempts. He was unable to label the RNA for various reasons (see Thesis).

3D reconstruction of ASPM RNA or ASPM protein in relation to the centrosome.

Failure is not an option

While he was unsuccessful in labeling RNAs around the centrosome using either proximity labeling technique, he did uncover unique mechanisms of localization of one RNA transcript to the centrosome – ASPM (Lo, Pearson, and Taliaferro 2024).

His work confirmed previous work that localization of ASPM RNA is co-translational. Yet surprisingly, ASPM RNA mislocalization did not mislocalize its encoded protein. Furthermore, he discovers that the ASPM RNA and ASPM protein reside in similar, yet distinct subcellular locales.

3D rendering of the predicted folding of an ASPM CDS isoform with AlphaFold 2. Colored by position across protein (N-terminus (blue), C-terminus (red).

Thinking outside the box

He also used in-silico AlphaFold predictions and computational approaches to predict localization elements common to other centrosome-localized transcripts (see Thesis).

While unpublished, he found that centrosome-localized RNAs have long coding sequences (CDS). These long CDS encode for long stretches of alpha-helices.

These two features likely serve two purposes for RNA localization: 1) the length of the CDS maintain the co-translational machinery intact for localization of the RNA transcript. 2) The alpha helices act as scaffolding to integrate into locomotive machinery or the coil-coil rich pericentrosomal matrix.

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