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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Special Emphasis Panel (ZRG1)
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Phillips, Andre W
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Cornell University
Schools of Arts and Sciences
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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
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:
Wakefield, Devin L; Holowka, David; Baird, Barbara (2017) The Fc?RI Signaling Cascade and Integrin Trafficking Converge at Patterned Ligand Surfaces. Mol Biol Cell :
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Holowka, David; Baird, Barbara (2016) Roles for lipid heterogeneity in immunoreceptor signaling. Biochim Biophys Acta 1861:830-836
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