SpLacZ-MERCS-Coupled CRISPRi Screening Identifies Novel Mitochondria-ER Contact Sites Regulators

Citation

Yang, Zheng (2025) SpLacZ-MERCS-Coupled CRISPRi Screening Identifies Novel Mitochondria-ER Contact Sites Regulators. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/r4m2-b064. https://resolver.caltech.edu/CaltechTHESIS:05012025-221548751

Abstract

Mitochondria-ER contact sites (MERCS) mark critical hotspots for a variety of cellular processes, including calcium homeostasis, lipid homeostasis, mitochondria dynamics, and quality control. Fluorescence-based tools have been the main approach to detect MERCS, with a large portion of studies using split fluorescent proteins, which assemble at sites of contact to yield a fluorescence signal. However, they have limitations, including little to no response to fluctuations in MERCS abundance, low sensitivity, and possible artifacts made due to reporter protein reconstitution. To overcome this, we developed the SpLacZ-MERCS sensor, the first MERCS reporter using split β-galactosidase (LacZ). Compared to using complementary GFP fragments that go to mitochondria and ER, SpLacZ-MERCS gives an integrated readout of MERCS activity for more accurate and quantitative monitoring of these contact sites in single cells over time. Our system has specific organelle targeting but does not induce artificial tethering, which allows it to be a standard tool for studying MERC dynamics in physiological and pathological conditions. Using pharmacological and genetic perturbations known to modulate mitochondria–ER interactions, we validated SpLacZ-MERCS as an effective and reliable sensor of MERCS abundance.

Beyond tool development, we sought to uncover the molecular mechanisms regulating MERCS using a genome-wide CRISPR interference (CRISPRi) screen combined with SpLacZ-MERCS. This unbiased approach led to the identification of RHOA, a small GTPase known for its roles in cytoskeletal dynamics and signal transduction as a novel regulator of MERCS. We found that RHOA directly interacts with the ER-resident protein VAPB and modulates its binding to PTPIP51, a mitochondrial protein involved in forming MERCS junctions. VAPB and PTPIP51 constitute a MERCS tethering complex. RHOA depletion or overexpression of CUL3 (which promotes RHOA degradation) results in reduced MERCS levels, while RHOA overexpression enhances MERCS formation. Notably, we discovered that disease-associated mutations in RHOA, CUL3, and VAPB—implicated in cancer, metabolic disorders, and neurodegeneration—disrupt MERCS regulation, suggesting a potential link between MERCS dysfunction and disease pathology.

Together, our study makes two significant contributions. SpLacZ-MERCS is a new signal-integrating MERCS reporter system that allows dynamic, cumulative tracking of mitochondria-ER interactions. RHOA has been established as a novel regulator of MERCS, providing a framework to understand how contact sites can be manipulated in a dynamic way upon cellular signals. These findings enhance the foundation of our understanding of MERCS regulation while also shedding light on new possible therapeutic targets for diseases associated with altered communication between mitochondria and the ER.

Thesis Files