Biology subjects

Hansen, S. D.

Publications and source records attributed to Hansen, S. D..

15 recordsLinked to original sources

bioRxiv2026

Single molecule Grb2 dynamics reveal spatiomechanical modulation of RTK signaling across length scales

Extracellular matrix (ECM)-mediated cell adhesion and tension regulate development and disease through crosstalk with Receptor Tyrosine Kinases (RTKs). RTKs stimulate proliferation, survival, and metabolic reprogramming via Ras/ERK and PI3K/Akt signaling. Emerging evidence identify the biophysical and biochemical integration of integrin-mediated adhesions with RTK signaling, however, the molecular mechanisms regulating this phenotype remain poorly understood. Here, we demonstrate that ECM stiffness potentiates ligand-mediated epidermal growth factor receptor (EGFR) signaling at scales ranging from the single adaptor protein, Grb2 upward to multicellular spheroids and tissues. We show that ECM stiffness-driven RTK signaling is mediated by integrin adhesions that reshape the membrane to induce the formation of RTK signaling hotspots distributed globally across the cell membrane. A parsimonious computational model and biochemical membrane perturbations causally implicate membrane curvature and topography in tension-dependent integrin-EGFR crosstalk. The results demonstrate how the mechanical properties of the ECM direct molecular recruitment and confinement of signaling molecules to regulate growth factor-dependent RTK signaling. Given that many RTK-driven solid tumors develop a stiff stroma, the findings could explain why many therapeutic tyrosine kinase inhibitors develop clinical resistance. The findings suggest clarifying how tension regulates transmembrane receptor signaling could identify unique force-dependent molecular regulators towards which new antitumor therapies could be applied.

biophysics↗

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↗

bioRxiv2026

Phosphorylation of the C-terminus of PI4KA inhibits lipid kinase activity

Phosphatidylinositol 4-kinase alpha (PI4KA) is a lipid kinase that generates phosphatidylinositol 4-phosphate (PI4P) from phosphatidylinositol (PI) at the plasma membrane (PM). PI4P generated by PI4KA is essential for both plasma membrane identity and for PIP2 and PIP3 signalling driven by the PLC and PI3K family of enzymes. While the structure of PI4KA is known, the regulatory mechanisms that control its activity are undefined. Here, we discovered PI4KA can be inhibited through phosphorylation of Y2090 in the kinase domain, which can be phosphorylated by multiple tyrosine kinases. Y2090 phosphorylation causes local conformational changes in the k12 C-terminal helix. PI4KA activity is predominantly controlled through membrane recruitment by EFR3, with Y2090 phosphorylation inhibiting EFR3 bound PI4KA. Phosphorylation of the k12 C-terminal helix is found in PI3Ks and PI4Ks, suggesting an evolutionarily conserved regulatory mechanism for diverse phosphoinositide kinases. Overall, our work reveals novel molecular insight into inhibitory post-translational regulation of PI4KA.

biochemistry↗

bioRxiv2026

Reconstitution of Ras-PI3Kγ membrane communication and feedback using light-induced signaling inputs

Biochemical reactions involving phosphatidylinositol phosphate (PIP) lipids and small GTPases serve essential roles in signal transduction immediately downstream of cell surface receptor activation. Interconnected positive and negative feedback loops link these distinct classes of reactions and allow for the rapid establishment of the steep and precisely oriented intracellular activity gradients that are hallmarks of cell polarity. Although genetic approaches have identified numerous molecules that potentially regulate feedback between GTPases and PIP lipids in cells, it is unclear how different types of feedback modulate the spatiotemporal dynamics of membrane signaling reactions. Here, we reconstitute communication and feedback between Ras GTPase and phosphatidylinositol 3-kinase gamma (PI3K{gamma})-mediated generation of PIP3 on supported membranes. We employ light-induced membrane recruitment of a guanine nucleotide exchange factor (GEF) to rapidly and reversibly shift steady-state conditions and monitor corresponding Ras activation and PIP3 production in time and space. Alone, the Ras-PI3K{gamma} module exhibits weak and transient activation due to global inhibitors. However, the introduction of GEF-mediated positive feedback drives local amplification of Ras activation and PIP3 generation, resulting in a wave of activity that propagates across the membrane surface. Spatial coupling between activated Ras and PIP3 molecules depends on their respective diffusion coefficients and the species involved in the feedback circuit. This work identifies activation thresholds, membrane diffusion, and feedback architecture as key factors that determine how small GTPases and PIP lipid modifying enzymes amplify membrane signaling reactions in the presence of global inhibitors.

biochemistry↗

bioRxiv2025

PIP4K attenuates PIP5K lipid kinase activity by disrupting membrane-mediated dimerization

The phosphatidylinositol 4-phosphate 5-kinase (PIP5K) family of enzymes generate most of the phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) lipids in eukaryotes. In solution, PIP5K exists in a weak monomer-dimer equilibrium but undergoes membrane-mediated dimerization, which potentiates lipid kinase activity. In vivo, however, PI(4,5)P2 levels are held remarkably constant due to the homeostatic regulation of PIP5K by PIP4K. We hypothesized that mechanisms that regulate PIP5K dimerization could function to buffer lipid kinase activity, thus providing a mechanism for maintaining relatively constant PI(4,5)P2 levels at the plasma membrane. Due the transient nature and density dependence of PIP5K dimerization, deciphering how other proteins modulate PIP5K dimerization has not been feasible. To address this limitation, we established a single molecule FRET assay to visualize membrane-mediated homodimerization and heterodimerization of PIP5K paralogs on supported lipid bilayers using Total Internal Reflection Fluorescence Microscopy (TIRF-M). Using this approach, we find that PIP4K attenuates PIP5K lipid kinase activity by disrupting membrane-mediated dimerization. Guided by structure prediction, we generated PIP4K mutants that are unable to disrupt PIP5K membrane-mediated dimerization thus preventing the attenuation lipid kinase activity. In vivo, mutations that disrupt the PIP4K-PIP5K interaction similarly prevent PIP4K-mediated inhibition of the PIP5K activity. Overall, this work reveals the molecular basis of the PIP4K-mediated inhibition of PIP5K, which underlies PI(4,5)P2 lipid homeostasis. Creation of this PIP5K dimerization FRET biosensor also establishes a novel tool for deciphering how proteins modulate membrane-mediated dimerization of PIP5K in the future.

biochemistry↗

bioRxiv2025

SH2-mediated steric occlusion of the C2 domain regulates autoinhibition of SHIP1 inositol 5-phosphatase

The Src homology 2 (SH2) domain containing inositol polyphosphate 5-phosphatase 1 (SHIP1) is an immune cell specific enzyme that regulates phosphatidylinositol-(3,4,5)-trisphosphate signaling at the plasma membrane following receptor activation. SHIP1 plays an important role in processes such as directed cell migration, endocytosis, and cortical membrane oscillations. Alterations in SHIP1 expression have been shown to perturb myeloid cell chemotaxis and differentiation. In the brain, SHIP1 regulate microglial cell behaviors, which has been linked to Alzheimers disease. Understanding the structural and functional relationships of SHIP1 is critical for developing ways to modulate SHIP1 membrane localization and lipid phosphatase activity during immune cell signaling. Recently, we discovered that the N-terminal SH2 domain of SHIP1 suppresses lipid phosphatase activity. SHIP1 autoinhibition can be relieved through interactions with receptor-derived phosphotyrosine (pY) peptides presented on membranes or in solution. Using hydrogen-deuterium exchange mass spectrometry (HDX-MS) we identified intramolecular contacts between the N-terminal SH2 domain and CBL1 motif of the C2 domain that limit SHIP1 membrane localization and activity. Single molecule measurements of purified SHIP1 on supported lipid bilayers and in neutrophil-like cells support a model in which the SH2 domain blocks membrane binding of the central catalytic module. Mutations that disrupt autoinhibition enhance the membrane binding frequency and increase the catalytic efficiency of SHIP1. Although dimerization of SHIP1 enhances membrane localization and the apparent phosphatase activity, it is not required for SHIP1 autoinhibition. Overall, our results provide new insight concerning SHIP1s structural organization, membrane binding dynamics, and the mechanism of autoinhibition.

biochemistry↗

bioRxiv2023

Molecular basis of product recognition during PIP5K-mediated production of PI(4,5)P2 with positive feedback

The ability for cells to localize and activate peripheral membrane binding proteins is critical for signal transduction. Ubiquitously important in these signaling processes in eukaryotic cells are phosphatidylinositol phosphate (PIP) lipids, which are dynamically phosphorylated by PIP lipid kinases on intracellular membranes. Functioning primarily at the plasma membrane, phosphatidylinositol-4-phosphate 5-kinases (PIP5K) catalyze the phosphorylation of PI(4)P to generate most PI(4,5)P2 lipids found in cells. Recently, we determined that PIP5K displays a positive feedback loop based on membrane-mediated dimerization and cooperative binding to its product, PI(4,5)P2. Here, we examine how two PIP5K motifs contribute to PI(4,5)P2 recognition to control membrane association and catalysis. Using a combination of single molecule TIRF microscopy and kinetic analysis of PI(4)P lipid phosphorylation, we map the sequence of steps that allow PIP5K to cooperatively engage PI(4,5)P2. We find that the specificity loop regulates the rate of PIP5K membrane association and helps orient the kinase to more effectively bind PIP lipids. Attaching the PIP5K specificity loop to other peripheral membrane binding proteins can enhance their membrane binding dynamics. After correctly orienting on the membrane, PIP5K transitions to binding PIP lipids in a structural motif previously referred to as the substrate or PIP binding motif (PIPBM). Our data reveals that the PIPBM has broad specificity for anionic lipids and serves a critical role in regulating membrane association in vitro and in vivo. The strength of the interaction between the PIPBM and PIP lipids depends on the membrane density and the extent phosphorylation on the inositol head group. We propose a two-step membrane binding model where the specificity loop and PIPBM act in concert to help PIP5K orient and productively engage anionic lipids to drive the positive feedback during PI(4,5)P2 production.

biochemistry↗

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↗

bioRxiv2023

Molecular dissection of PI3Kβ synergistic activation by receptor tyrosine kinases, GβGγ, and Rho-family GTPases

The class 1A phosphoinositide 3-kinase (PI3K) beta (PI3K{beta}) is functionally unique in the ability to integrate signals derived from receptor tyrosine kinases (RTKs), heterotrimeric guanine nucleotide-binding protein (G-protein)-coupled receptors (GPCRs), and Rho-family GTPases. The mechanism by which PI3K{beta} prioritizes interactions with various membrane tethered signaling inputs, however, remains unclear. Previous experiments have not been able to elucidate whether interactions with membrane-tethered proteins primarily control PI3K{beta} localization versus directly modulate lipid kinase activity. To address this gap in our understanding of PI3K{beta} regulation, we established an assay to directly visualize and decipher how three distinct protein interactions regulate PI3K{beta} when presented to the kinase in a biologically relevant configuration on supported lipid bilayers. Using single molecule Total Internal Reflection Fluorescence (TIRF) Microscopy, we determined the mechanism controlling membrane localization of PI3K{beta}, prioritization of signaling inputs, and lipid kinase activation. We find that auto-inhibited PI3K{beta} prioritizes interactions with RTK-derived tyrosine phosphorylated (pY) peptides before engaging either G{beta}G{gamma} or Rac1(GTP). Although pY peptides strongly localize PI3K{beta} to membranes, stimulation of lipid kinase activity is modest. In the presence of either pY/G{beta}G{gamma} or pY/Rac1(GTP), PI3K{beta} activity is dramatically enhanced beyond what can be explained by simply increasing the strength of membrane localization. Instead, PI3K{beta} is synergistically activated by pY/G{beta}G{gamma} and pY/Rac1(GTP) through a mechanism consistent with allosteric regulation.

biochemistry↗

bioRxiv2023

Allosteric activation or inhibition of PI3Kγ mediated through conformational changes in the p110γ helical domain

PI3K{gamma} is a critical immune signaling enzyme activated downstream of diverse cell surface molecules, including Ras, PKC{beta} activated by the IgE receptor, and G{beta}{gamma} subunits released from activated GPCRs. PI3K{gamma} can form two distinct complexes, with the p110{gamma} catalytic subunit binding to either a p101 or p84 regulatory subunit, with these complexes being differentially activated by upstream stimuli. Here using a combination of cryo electron microscopy, HDX-MS, and biochemical assays we have identified novel roles of the helical domain of p110{gamma} in regulating lipid kinase activity of distinct PI3K{gamma} complexes. We defined the molecular basis for how an allosteric inhibitory nanobody potently inhibits kinase activity through rigidifying the helical domain and regulatory motif of the kinase domain. The nanobody did not block either p110{gamma} membrane recruitment or Ras/G{beta}{gamma} binding, but instead decreased ATP turnover. We also identified that p110{gamma} can be activated by dual PKC{beta} helical domain phosphorylation leading to partial unfolding of an N-terminal region of the helical domain. PKC{beta} phosphorylation is selective for p110{gamma}-p84 compared to p110{gamma}-p101, driven by differential dynamics of the helical domain of these different complexes. Nanobody binding prevented PKC{beta} mediated phosphorylation. Overall, this works shows an unexpected allosteric regulatory role of the helical domain of p110{gamma} that is distinct between p110{gamma}-p84 and p110{gamma}-p101 and reveals how this can be modulated by either phosphorylation or allosteric inhibitory binding partners. This opens possibilities of future allosteric inhibitor development for therapeutic intervention.

biochemistry↗

bioRxiv2022

A Novel Homeostatic Mechanism Tunes PI(4,5)P2-dependent Signaling at the Plasma Membrane

The lipid molecule phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2) controls all aspects of plasma membrane (PM) function in animal cells, from its selective permeability to the attachment of the cytoskeleton. Although disruption of PI(4,5)P2 is associated with a wide range of diseases, it remains unclear how cells sense and maintain PI(4,5)P2 levels to support various cell functions. Here, we show that the PIP4K family of enzymes that synthesize PI(4,5)P2 via a minor pathway, also function as sensors of tonic PI(4,5)P2 levels. PIP4Ks are recruited to the PM by elevated PI(4,5)P2 levels, where they inhibit the major PI(4,5)P2-synthesizing PIP5Ks. Perturbation of this simple homeostatic mechanism reveals differential sensitivity of PI(4,5)P2-dependent signaling to elevated PI(4,5)P2 levels. These findings reveal that a subset of PI(4,5)P2-driven functions may drive disease associated with disrupted PI(4,5)P2 homeostasis. One-Sentence SummaryThe enzyme PIP4K functions as both a sensor and negative regulator of PI(4,5)P2 synthesis by the closely related PIP5K enzymes, tuning the activity of numerous membrane functions.

cell biology↗

bioRxiv2021

Membrane-mediated dimerization potentiates PIP5K lipid kinase activity

The phosphatidylinositol 4-phosphate 5-kinase (PIP5K) family of lipid modifying enzymes generate the majority of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) lipids found at the plasma membrane in eukaryotic cells. PI(4,5)P2 lipids serve a critical role in regulating receptor activation, ion channel gating, endocytosis, and actin nucleation. Here we describe how PIP5K activity is regulated by cooperative binding to PI(4,5)P2 lipids and membrane-mediated dimerization of the kinase domain. In contrast to constitutively dimeric phosphatidylinositol 5-phosphate 4-kinase (PIP4K, type II PIPK), solution PIP5K exists in a weak monomer-dimer equilibrium. PIP5K monomers can associate with PI(4,5)P2 containing membranes and dimerize in a protein density dependent manner. Although dispensable for PI(4,5)P2 binding and lipid kinase activity, dimerization enhances the catalytic efficiency of PIP5K through a mechanism consistent with allosteric regulation. Additionally, dimerization amplifies stochastic variation in the kinase reaction velocity and strengthens effects such as the recently described stochastic geometry sensing. Overall, the mechanism of PIP5K membrane binding creates a broad dynamic range of lipid kinase activities that are coupled to the density of PI(4,5)P2 and membrane bound kinase.

biochemistry↗

bioRxiv2021

Structure of the phosphoinositide 3-kinase p110γ-p101 complex reveals molecular mechanism of GPCR activation

The class IB phosphoinositide 3-kinase (PI3K), PI3K{gamma}, is a master regulator of immune cell function, and a promising drug target for both cancer and inflammatory diseases. Critical to PI3K{gamma} function is the association of the p110{gamma} catalytic subunit to either a p101 or p84 regulatory subunit, which mediates activation by G-protein coupled receptors (GPCRs). Here, we report the cryo-EM structure of a heterodimeric PI3K{gamma} complex, p110{gamma}-p101. This structure reveals a unique assembly of catalytic and regulatory subunits that is distinct from other class I PI3K complexes. p101 mediates activation through its G{beta}{gamma} binding domain, recruiting the heterodimer to the membrane and allowing for engagement of a secondary G{beta}{gamma} binding site in p110{gamma}. Multiple oncogenic mutations mapped to these novel interfaces and enhanced G{beta}{gamma} activation. A nanobody that specifically binds to the p101-G{beta}{gamma} interface blocks activation providing a novel tool to study and target p110{gamma}-p101-specific signaling events in vivo.

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↗

bioRxiv2017

Bacterial Microtubules Exhibit Polarized Growth, Mixed-Polarity Bundling, and Destabilization by GTP Hydrolysis

Bacteria of the genus Prosthecobacter express homologs of eukaryotic -and {beta}-tubulin, called BtubA and BtubB, that have been observed to assemble into bacterial microtubules (bMTs). The btubAB genes likely entered the Prosthecobacter lineage via horizontal gene transfer and may derive from an early ancestor of the modern eukaryotic microtubule (MT). Previous biochemical studies revealed that BtubA/B polymerization is GTP-dependent and reversible and that BtubA/B folding does not require chaperones. To better understand bMT behavior and gain insight into the evolution of microtubule dynamics, we characterized in vitro bMT assembly using a combination of polymerization kinetics assays, and microscopy. Like eukaryotic microtubules, bMTs exhibit polarized growth with different assembly rates at each end. GTP hydrolysis stimulated by bMT polymerization drives a stochastic mechanism of bMT disassembly that occurs via polymer breakage. We also observed treadmilling (continuous addition and loss of subunits at opposite ends) of bMT fragments. Unlike MTs, polymerization of bMTs requires KCl, which reduces the critical concentration for BtubA/B assembly and induces bMTs to form stable mixed-orientation bundles in the absence of any additional bMT-binding proteins. Our results suggest that at potassium concentrations resembling that inside the cytoplasm of Prosthecobacter, bMT stabilization through self-association may be a default behavior. The complex dynamics we observe in both stabilized and unstabilized bMTs may reflect common properties of an ancestral eukaryotic tubulin polymer.\n\nImportanceMicrotubules are polymers within all eukaryotic cells that perform critical functions: they segregate chromosomes in cell division, organize intracellular transport by serving as tracks for molecular motors, and support the flagella that allow sperm to swim. These functions rely on microtubules remarkable range of tunable dynamic behaviors. Recently discovered bacterial microtubules composed of an evolutionarily related protein are evolved from a missing link in microtubule evolution, the ancestral eukaryotic tubulin polymer. Using microscopy and biochemical approaches to characterize bacterial microtubules, we observed that they exhibit complex and structurally polarized dynamic behavior like eukaryotic microtubules, but differ in how they self-associate into bundles and become destabilized. Our results demonstrate the diversity of mechanisms that microtubule-like filaments employ to promote filament dynamics and monomer turnover.

microbiology↗