Biology subjects

Waddell, G. L.

Publications and source records attributed to Waddell, G. L..

3 recordsLinked to original sources

bioRxiv2026

SHIP1 partitions between cortical oscillations and membrane puncta to regulate neutrophil chemotaxis

As frontline defenders in the innate immune system, neutrophils traffic to sites of infection by detecting chemical gradients, polarizing, and migrating towards foreign pathogens. This requires communication between small GTPases, phosphatidylinositol phosphate (PIP) lipids, and the actin cytoskeleton to generate forces that drive leading-edge membrane protrusions. Central to the coordination of these signaling events is the hematopoietic-cell-specific 5-inositol lipid phosphatase SHIP1 (or INPP5D), which shapes PIP lipid domains by dephosphorylating phosphatidylinositol-(3,4,5)-trisphosphate (PIP3) to generate PI(3,4)P2. To decipher mechanisms controlling SHIP1 membrane localization during neutrophil chemotaxis, we combined genetic manipulation of SHIP1 with live cell Total Internal Reflection Fluorescence (TIRF) microscopy. Our results reveal two modes of SHIP1 localization: (1) cortical oscillations regulated by the membrane curvature sensing protein FBP17, and (2) dynamic membrane puncta organized by the adaptor protein Nck1. Localization to both membrane structures requires the proline-rich C-terminal domain (CTD) of SHIP1. Neutrophil-differentiated HL60 cells solely expressing a cytoplasmic localized SHIP1 CTD deletion mutant exhibit chemotaxis defects that phenocopy complete SHIP1 loss of function. Overall, this work reveals a dual mechanism by which SHIP1 integrates signals from membrane curvature sensing and adaptor proteins to control neutrophil cell migration.

cell biology↗

bioRxiv2023

Mechanisms controlling membrane recruitment and activation of autoinhibited SHIP1

Signal transduction downstream of growth factor and immune receptor activation relies on the production of phosphatidylinositol-(3,4,5)-trisphosphate (PI(3,4,5)P3) lipids by phosphoinositide-3-kinase (PI3K). Regulating the strength and duration of PI3K signaling in immune cells, Src homology 2 domain-containing inositol 5-phosphatase 1 (SHIP1) controls the dephosphorylation of PI(3,4,5)P3 to generate PI(3,4)P2. Although SHIP1 has been shown to regulate neutrophil chemotaxis, B-cell signaling, and cortical oscillations in mast cells, the role that lipid and protein interactions serve in controlling SHIP1 membrane recruitment and activity remains unclear. Using single molecule TIRF microscopy, we directly visualized membrane recruitment and activation of SHIP1 on supported lipid bilayers and the cellular plasma membrane. We find that SHIP1s interactions with lipids are insensitive to dynamic changes in PI(3,4,5)P3 both in vitro and in vivo. Very transient SHIP1 membrane interactions were detected only when membranes contained a combination of phosphatidylserine (PS) and PI(3,4,5)P3 lipids. Molecular dissection reveals that SHIP1 is autoinhibited with the N-terminal SH2 domain playing a critical role in suppressing phosphatase activity. Robust SHIP1 membrane localization and relief of autoinhibition can be achieved through interactions with immunoreceptor derived phosphopeptides presented either in solution or conjugated to supported membranes. Overall, this work provides new mechanistic details concerning the dynamic interplay between lipid binding specificity, protein-protein interactions, and activation of autoinhibited SHIP1.

biochemistry↗

bioRxiv2019

Rapid and Inexpensive Preparation of Genome-Wide Nucleosome Footprints from Model and Non-Model Organisms

Eukaryotic DNA is packaged into nucleosomes, the smallest repeating unit of chromatin. The positions of nucleosomes determine the relative accessibility of genomic DNA. Several protocols exist for mapping nucleosome positions in eukaryotic genomes in order to study the relationship between chromatin structure and DNA-dependent processes. These nucleosome mapping protocols can be laborious and, at minimum, require two to three days to isolate nucleosome-protected DNA fragments. We have developed a streamlined protocol for mapping nucleosomes from S. cerevisiae liquid culture or from patches on solid agar. This method isolates nucleosome-sized footprints in three hours using 1.5 ml tubes with minimal chemical waste. We validate that these footprints match those produced by previously published methods and we demonstrate that our protocol works for N. crassa and S. pombe. A slightly modified protocol can be used for isolation of nucleosome-protected DNA fragments from a variety of wild fungal specimens thereby providing a simple, easily multiplexed and unified strategy to map nucleosome positions in model and non-model fungi. Finally, we demonstrate recovery of nucleosome footprints from the diploid myeloid leukemia cell line PLB-985 in less than three hours using an abbreviated version of the same protocol. With reduced volume and incubation times and a streamlined workflow, the described method should be compatible with high-throughput, automated creation of MNase-seq libraries. We believe this simple validated method for rapidly producing sequencing-ready nucleosome footprints from a variety of organisms will make nucleosome mapping studies widely accessible to researchers globally.

genetics↗