This continuing research is aimed at characterizing, at micro- and nanoscales, the collective molecular events of cellular signaling that are initiated by plasma membrane receptors or ion channels and are highly orchestrated in space and time. Mast cells, which are stimulated by IgE-receptors and play a pivotal role in immune responses, serve as a valuable model system for investigating basic mechanisms by which cells respond to specific stimuli in a noisy environment. Proposed research will build on established thrusts: 1) Delineate with increasing resolution the initiation of stimulated cell signaling with a focus on critical but subtle events occurring at cellular membranes; 2) Translate the advanced technologies and refined hypotheses we have developed in model cells to disruption of cellular processes associated with neurodegenerative diseases. Innovative aspects include advancing an imaging modality of fluorescence correlation spectroscopy (ImFCS) to measure dynamic interactions of signaling-related components within the heterogeneous plasma membrane. The large data sets and robust analytics afforded by ImFCS yield precise values for multi-component diffusion and transient confinement parameters, revealing subtle interactions experienced by selective probes. This and other quantitative fluorescence microscopies will be used to evaluate constitutive and stimulated formation of kinase complexes, membrane trafficking, and mitochondrial dynamics, which are disrupted in cellular pathologies.
Specific Aim 1 will implement ImFCS to measure diffusion properties of transmembrane proteins and proteins anchored to plasma membrane inner and outer leaflets to reveal distinctive environments experienced by these probes before and after antigen-crosslinking of IgE-receptors to initiate transmembrane signaling. We will measure stimulated changes in seconds timescale and collaboratively adapt theoretical models to interpret our results in terms of time-dependent changes of IgE-Fc?RI cluster size as related to early signaling events.
Specific Aim 2 will collaboratively investigate model cells to gain mechanistic insight into cell- based pathologies associated with neurodegeneration. Building on established phenotypes, we will continue to evaluate structural features of ?-synuclein variants that disruptively modulate membrane trafficking and mitochondrial activities in Parkinson?s disease. We will use micro-patterned surfaces and ImFCS to yield new understanding of microglia-mediated hydrolysis of A? fibrils that goes awry in Alzheimer?s disease and phosphatidyl inositol kinase complexes that are disrupted in hypomyelination diseases.

Public Health Relevance

The heterogeneous plasma membrane milieu of eukaryotic cells maintains a steady state of protein and lipid interactions that support basal cell function and, while serving as a selective barrier, is poised to respond appropriately to environmental stimuli. A primary goal of our continuing studies is molecular level elucidation of the structural interactions occurring within the dynamic plasma membrane that are altered by antigen crosslinking of IgE-Receptors to initiate intracellular signaling cascades in the allergic immune response. We will also engage in highly collaborative studies to translate insight and high-resolution approaches gained from detailed characterization of this model cell system to investigate membrane interactions that participate in neurological cell activities and are disrupted in neurodegenerative diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM117552-36
Application #
9838516
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Melillo, Amanda A
Project Start
1981-08-01
Project End
2023-12-31
Budget Start
2020-01-01
Budget End
2020-12-31
Support Year
36
Fiscal Year
2020
Total Cost
Indirect Cost
Name
Cornell University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
872612445
City
Ithaca
State
NY
Country
United States
Zip Code
14850
Holowka, David; Thanapuasuwan, Kankanit; Baird, Barbara (2018) Short chain ceramides disrupt immunoreceptor signaling by inhibiting segregation of Lo from Ld Plasma membrane components. Biol Open 7:
Mitra, Eshan D; Whitehead, Samuel C; Holowka, David et al. (2018) Computation of a Theoretical Membrane Phase Diagram and the Role of Phase in Lipid-Raft-Mediated Protein Organization. J Phys Chem B 122:3500-3513
Wakefield, Devin L; Holowka, David; Baird, Barbara (2017) The Fc?RI Signaling Cascade and Integrin Trafficking Converge at Patterned Ligand Surfaces. Mol Biol Cell :
Holowka, David; Baird, Barbara (2017) Mechanisms of epidermal growth factor receptor signaling as characterized by patterned ligand activation and mutational analysis. Biochim Biophys Acta Biomembr 1859:1430-1435
Korzeniowski, Marek K; Wisniewski, Eva; Baird, Barbara et al. (2017) Molecular anatomy of the early events in STIM1 activation - oligomerization or conformational change? J Cell Sci 130:2821-2832
Holowka, David; Baird, Barbara (2016) Roles for lipid heterogeneity in immunoreceptor signaling. Biochim Biophys Acta 1861:830-836
Wilson, Joshua D; Shelby, Sarah A; Holowka, David et al. (2016) Rab11 Regulates the Mast Cell Exocytic Response. Traffic 17:1027-41
Shelby, Sarah A; Veatch, Sarah L; Holowka, David A et al. (2016) Functional nanoscale coupling of Lyn kinase with IgE-Fc?RI is restricted by the actin cytoskeleton in early antigen-stimulated signaling. Mol Biol Cell 27:3645-3658
Sun, Chao; Wakefield, Devin L; Han, Yimo et al. (2016) Graphene Oxide Nanosheets Stimulate Ruffling and Shedding of Mammalian Cell Plasma Membranes. Chem 1:273-286