Graduate Research Assistant, UW-Madison
BIOGRAPHY
SEP 13, 2023 9:00 AM PDT
Gibco Cell Culture Heroes - Guy Kunzmann - Investigating the conditionally essential role of protein UFMylation in proliferating human cells
Speaker
Event Date & Time
Date: September 13, 2023
Time: 9:00am PST, 12:00pm EST
Abstract
CRISPR-based forward genetic screens can be used to identify essential genes in proliferating human cell lines. Although environmental factors also influence gene essentiality, most CRISPR-based screens are performed on cultured cells growing in conventional media that poorly reflect nutrient conditions within the human body. Previously, we developed the first systematically designed physiologic cell culture medium, Human Plasma-Like Medium (HPLM), which contains over 70 defined components at concentrations that more closely reflect those in human blood. To test the hypothesis that medium composition can impact gene essentiality we performed genome-wide CRISPR screens on the K562 chronic myelogenous leukemia cell line grown in either HPLM or RPMI – the conventional medium used to culture blood cells. Analysis of our screen results revealed hundreds of conditionally essential genes that collectively span diverse biological processes. Among the RPMI-essential hits we identified were components required for UFMylation, a system that attaches ubiquitin-fold modifier 1 (UFM1) to target proteins. UFMylation is a ubiquitin-like PTM that is best characterized for its role regulating selective autophagy of the ER through modification of ribosomal subunit RPL26 (termed ER-phagy).
To validate our screen results, we first confirmed that UFM1-knockout K562 cells show a conditional growth defect in RPMI versus HPLM. We then traced these effects to alanine, a component uniquely defined in HPLM but absent from RPMI. Deletion of UFM1 lead to a decrease in most amino acids when cells were grown in RPMI. However, it is unclear why alanine, a non-essential amino acid, can support amino acid abundance and cell growth in UFM1-knockout cells. Guided by these preliminary data, we are continuing to investigate the contribution of protein UFMylation to human cell fitness and why the importance of this PTM depends on alanine availability.
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Learning Objectives
- in vitro culture conditional alters human cell physiology
- UFMylation is a conditionally essential post-translational modification
- Identification of a link between alanine availability and UFMylation
Webinars will be available for unlimited on-demand viewing after live event.
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