Utilizing Fluorescence Microscopy to Characterize the Subcellular Distribution of the Novel Protein Acheron

Authors

Varun Sheel, University of Massachusetts AmherstFollow

ORCID

https://orcid.org/0000-0002-5363-8118

Access Type

Open Access Thesis

Document Type

thesis

Degree Program

Molecular & Cellular Biology

Degree Type

Master of Science (M.S.)

Year Degree Awarded

2021

Month Degree Awarded

September

Abstract

All cells carry the genetic machinery required to commit cell suicide; a process known as programmed cell death (PCD). While the ability to initiate PCD serves a number of useful purposes during development and homeostasis, misregulation of PCD is the underlying basis of most human diseases, including cancer, autoimmunity disorders and neurodegeneration. Using the tobacco hawkmoth Manduca sexta as a model organism, the Schwartz lab at UMass has demonstrated that PCD requires de novo gene expression and has cloned many death-associated genes. One of these genes encodes a novel protein that was named Acheron after one of the rivers of the Underworld in Greek mythology. Acheron (also known as Lupus Antigen Related Protein 6; Larp6) is an RNA binding protein that mediates a number of cellular processes, including cell survival, angiogenesis, migration, and differentiation. The molecular mechanisms that mediate Acheron’s diverse roles are poorly understood, but several lines of evidence suggest that it is mediated in part by protein protein interactions and motifs that target it to different cellular compartments. In this thesis, I employed immunofluorescence and confocal microscopy to conduct two studies employing mammalian cells. The first study was to determine the subcellular localization for Acheron in normal cells and cells treated with growth factors. I found that Acheron predominantly coincides with the microtubule cytoskeleton. In our second study, I tested the hypothesis that Acheron’s binding partners BAD, Human Homolog of Ariadne-1 (HHARI) and Calcium/Calmodulin-dependent Serine Protein Kinase (CASK) colocalize with Acheron. As part of this analysis, I sought to determine if Acheron could facilitate the translocation of CASK to the nucleus. I found that Acheron localizes predominantly to microtubules, with some expression in the cytoplasm and nucleus. When Acheron and its partners are co-labeled in cycling mouse C₂C₁₂ myoblasts or human U2-OS osteosarcoma cells, Acheron did not colocalize with BAD, HHARI or CASK on microtubules. However, I found that when Acheron is driven into the nucleus with high levels of growth factors, CASK also appeared to translocate to both the nucleus and to microtubules, where it colocalized with Acheron. The data acquired through these studies should provide not only insights into the subcellular distribution of Acheron and its potential binding partners but may also help elucidate its roles in programmed cell death, differentiation, and pathogenesis in mammalian models.

DOI

https://doi.org/10.7275/23719571.0

First Advisor

Lawrence M. Schwartz

Second Advisor

Rolf O. Karlstrom

Third Advisor

Thomas J. Maresca

Creative Commons License

This work is licensed under a Creative Commons Attribution 4.0 License.

Recommended Citation

Sheel, Varun, "Utilizing Fluorescence Microscopy to Characterize the Subcellular Distribution of the Novel Protein Acheron" (2021). Masters Theses. 1115.
https://doi.org/10.7275/23719571.0 https://scholarworks.umass.edu/masters_theses_2/1115