# In-situ architecture of membrane contact sites mediating organelle fission

> **NIH NIH DP2** · UNIVERSITY OF MICHIGAN AT ANN ARBOR · 2022 · $1,382,214

## Abstract

PROJECT SUMMARY
Sophisticated compartmentalization into membrane enclosed organelles of dedicated function is a hallmark of
eukaryotic cells. Organelles come in close apposition to each other forming membrane contact sites that are
nodes for communication, critical for many cellular functions. Despite their ubiquitous presence, little remains
known about how the molecular components of these contact organize into functional signaling conduits between
disparate organelles. This is largely because the dynamic nature and low cellular copy number of these contact
sites make it impossible to use conventional biochemical approaches to purify them and embark on classical
structure-function studies using X-crystallography, NMR, and single particle cryo-electron microscopy. Here, we
propose integration of novel cutting-edge microscopy modalities to visualize organellar interactions directly within
unperturbed cellular context. We will establish our workflow on one contact site formed by proteins on
endoplasmic reticulum, mitochondria, and lysosomes, respectively. Proteins at this contact site will be tagged
within iPSCs, which will be differentiated into neurons on microscopy grids. Confocal microscopy at cryogenic
temperatures will enable detection of these contacts with high subcellular precision. Guided by the fluorescence
localization, thin electron-transparent windows will be micromachined inside these cells. Subsequent cryo-
electron tomography, subtomogram averaging and deep-classification will enable three-dimensional structure
determination of these contact sites. Our work will help answer a vital biological question: how ER-mitochondria-
lysosome contact site marks mitochondria for fission, a process essential for maintaining their healthy supply in
the cell. The pipelines for automated sample preparation, data collection, and processing established during this
project will serve as a transformative blueprint for future structural studies in-situ. This project will lay a foundation
towards high-resolution structural characterization of the emerging, underexplored and complex field of
membrane contact sites and a mechanistic basis for understanding their function in health and disease. This
project will provide much needed directional framework to researchers elsewhere who are interested in studying
protein structures directly in cells, isolated tissues and organoids.

## Key facts

- **NIH application ID:** 10472370
- **Project number:** 1DP2GM150019-01
- **Recipient organization:** UNIVERSITY OF MICHIGAN AT ANN ARBOR
- **Principal Investigator:** Shyamal Mosalaganti
- **Activity code:** DP2 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $1,382,214
- **Award type:** 1
- **Project period:** 2022-09-01 → 2025-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10472370

## Citation

> US National Institutes of Health, RePORTER application 10472370, In-situ architecture of membrane contact sites mediating organelle fission (1DP2GM150019-01). Retrieved via AI Analytics 2026-05-27 from https://api.ai-analytics.org/grant/nih/10472370. Licensed CC0.

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