# Noncanonical regulatory mechanisms in cell biology

> **NIH NIH R35** · YALE UNIVERSITY · 2023 · $598,714

## Abstract

Project summary: This project expands on decades of success elucidating the genetic and molecular
underpinnings of intercellular communication and cytoskeletal remodeling in gamete development in females
and males. We propose research in two interesting areas: 1) the inhibition of cellular abscission during
cytokinesis of dividing germline cells, and 2) the molecular regulation and functional significance of highly
efficient tissue-specific stop codon readthrough during protein translation. Proliferating germline cells in animal
ovaries and testes characteristically fail to complete abscission, leading to cell clusters that remain connected
by intercellular bridges, called ring canals. Incomplete cytokinesis also occurs in several lymphoproliferative
disorders, highlighting the importance of understanding how this noncanonical endpoint to mitosis is controlled.
Using live imaging of germline mitosis in the Drosophila testis, we discovered a previously unknown
intermediate step in ring canal formation involving a midbody-like structure that remodels into a channel
between daughter cells. To learn how this maturation step occurs and how the molecular machinery that drives
abscission is inhibited from localizing to midbodies, we will use localized biotinylation to identify midbody
components and probe the genetic requirement for proteins known to function during cytokinesis. We will also
use localized biotinlylation to identify ring canal proteins at the plasma membrane-cytoskeleton interface. Our
interest in stop codon readthrough stems from our extensive analysis of the kelch gene and its function during
oogenesis. The kelch mRNA encodes a large open reading frame (ORF) punctuated by a single stop codon. In
ovaries, translation terminates at the stop to produce a ring canal protein and there is no apparent function for
the second ORF. In contrast, we have observed remarkably high efficiency stop codon readthrough in nerves
of the central nervous system (CNS) of larvae and adults, producing abundant ORF1+ORF2 protein.
Furthermore, kelch and several other genes in Drosophila display efficient stop codon readthrough specifically
in the CNS, suggesting the presence of many proteins with carboxy-terminal extensions of unknown function.
We will systematically analyze the scope and scale of stop codon readthrough using ribosome profiling of
neuronal tissue and mass spectrometry of total protein lysates to identify readthrough peptides. To define the
mechanism of readthrough, we will use genetic screens to investigate both stimulatory cis-acting sequences
flanking stop codons in readthrough genes and trans-acting factors. Finally, to understand the function of
neuronal stop codon readthrough, we will use gene editing to ablate ORF2 from kelch and several other genes
and carefully analyze phenotypes using cell biological and behavioral assays. This research program will lead
to discoveries concerning the fundamental cell biological and genetic mechanisms t...

## Key facts

- **NIH application ID:** 10616490
- **Project number:** 5R35GM141961-03
- **Recipient organization:** YALE UNIVERSITY
- **Principal Investigator:** Lynn COOLEY
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $598,714
- **Award type:** 5
- **Project period:** 2021-05-01 → 2026-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10616490

## Citation

> US National Institutes of Health, RePORTER application 10616490, Noncanonical regulatory mechanisms in cell biology (5R35GM141961-03). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10616490. Licensed CC0.

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