# Function and assembly of toxin-stabilized domains

> **NIH NIH R01** · UNIVERSITY OF VIRGINIA · 2020 · $369,937

## Abstract

Lipids and proteins form a variety of complexes and domains in cellular membranes. The underlying
principles that govern the assembly and function of these structures remain enigmatic. To address this
gap in knowledge, we have focused on critically testing key predictions of a prevalent model of
membrane domain organization: the lipid raft hypothesis. Current models propose that raft domains
normally exist as nanoscale compositional fluctuations at steady state in cells, but can be stabilized by
proteins to form functional rafts. The non-toxic membrane binding subunit of cholera toxin (CTxB) is an
example of a protein that can cluster raft-associated glycolipids to assembled stabilized raft domains.
In the previous funding period, we examined the mechanisms by which toxin-stabilized domains form
and asked whether they function to mechanically deform cell membranes to facilitate CTxB uptake by
clathrin independent endocytosis. We tested this using a novel variant of CTxB that is unable to cluster
glycolipids. Our results led to the unexpected discover that microtubules, dynein, and dynactin
generate pulling forces at sites of clathrin independent uptake of CTxB. Our goal for the upcoming
funding period is to better understand how microtubules and dynein/dynactin participate in clathrin-
independent endocytosis and how CTxB is selectively sorted into these specialized clathrin-
independent pathways. Using a combination of cell biological and live cell imaging approaches, we will
tackle these questions through three specific aims. First, we will determine if stabilized rafts sort CTxB
into clathrin independent endocytic pathways. Second, we will test the hypothesis that multiple clathrin-
independent pathways exploit microtubules and motors to tubulate and scission the plasma membrane.
Finally, we will identify cellular machinery responsible for linking microtubules and dynein/dynactin to
nascent clathrin-independent carriers. Successful completion of this work will provide fundamental
insights into the functions of stabilized rafts and uncover new mechanism by which toxins manipulate
and hijack cellular machinery for their own purposes. It will also advance our understanding of how
microtubules and microtubule-associated motors function in membrane trafficking events and reveal
novel role(s) for microtubules, dynein, and dynactin at the plasma membrane. Finally, it will define new
mechanisms and machinery that contribute to the assembly of clathrin independent endocytic carriers.

## Key facts

- **NIH application ID:** 9925238
- **Project number:** 5R01GM106720-09
- **Recipient organization:** UNIVERSITY OF VIRGINIA
- **Principal Investigator:** Anne K Kenworthy
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $369,937
- **Award type:** 5
- **Project period:** 2013-07-15 → 2022-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9925238

## Citation

> US National Institutes of Health, RePORTER application 9925238, Function and assembly of toxin-stabilized domains (5R01GM106720-09). Retrieved via AI Analytics 2026-05-27 from https://api.ai-analytics.org/grant/nih/9925238. Licensed CC0.

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