Research

Current Projects

1. Delineating subtle heterogeneity in plasma membranes of resting cells, which is modulated upon stimulation to mediate transmembrane signaling.

A. Imaging Fluorescence Correlation Spectroscopy (ImFCS) to measure precise diffusion coefficients and reveal changes in membrane organization occurring with antigen-activation of IgE-FcεRI

B. Microvillar Cartography (MC) to Investigate Control of IgE-FcεRI Signaling by Membrane Topography

2. Elucidating mechanisms by which alpha-synuclein perturbs cellular functions, as related to Parkinson’s disease and other neurodegenerative diseases.

Movie – Antigen binding to sensitized mast cells stimulates reorganization of actin cytoskeleton and exocytosis of intracellular vesicles. IgE specific for antigen bind to FcεRI receptors on mast cells and thereby sensitize cell to this antigen. Total internal reflection fluorescence (TIRF) microscopy: F-Actin cytoskeleton is labeled with LifeAct-mApple, red fluorescence. Intracellular vesicles are labeled VAMP8-pHl, green fluorescence that is dramatically enhanced when vesicles are exocytosed and exposed to extracellular medium at higher pH. This movie begins with unstimulated cells, and stimulating antigen is added at ~30 seconds. Movie from Wilson, Shelby, Holowka, Baird “Rab11 Regulates the Mast Cell Exocytic Response” Traffic 17(9): 1027-1041 (2016).

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Recently Completed Projects

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Summary of Research Contributions

  • Plasma membrane heterogeneity and roles in regulating and targeting cellular signaling
  • Quantitative fluorescence spectroscopy and microscopy to investigate structural interactions
  • Nanoscale-resolution microscopy and micro/nano fabrication to investigate spatio-temporally controlled assemblies
  • Fundamental parameters of ligands binding to cell surface receptors
  • Mechanisms by which early assemblies of signaling components link to intracellular response processes
  • Using synthetic materials to interrogate cellular structures and processes

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Plasma membrane heterogeneity and roles in regulating and targeting cellular signaling:

Our research group has been a major player in characterizing plasma membrane heterogeneity, now widely recognized to play important roles in regulating and targeting cellular signaling. We were the first to show that antigen-crosslinking causes lipid-mediated coupling of immune receptors with their cognate kinases in the first transmembrane signaling event. We continue to advance efforts toward rigorous characterization of such membrane domains in terms of the dynamic interactions of membrane lipids and proteins that link to the cytoskeleton in living cells.

Selected Publications:

Field, K.A., D. Holowka and B. Baird: Compartmentalized activation of the high affinity IgE receptor within membrane domains.  J. Biol. Chem. 272: 4276-4280, 1997.

Young, R.M., X. Zheng, D. Holowka and B. Baird: Reconstitution of Regulated Phosphorylation of FceRI by a Lipid Raft-excluded Protein-tyrosine Phosphatase. J. Biol. Chem. 280(2): 1230-1235, 2005.

Baumgart, T., A.T. Hammond, P. Sengupta, S.T. Hess, D.A. Holowka, B.A. Baird and W.W. Webb: Large-scale fluid/fluid phase separation of proteins and lipids in giant plasma membrane vesicles.  Proc. Natl. Acad. Sci. USA 104(9): 3165-3170, 2007.

Veatch, S., P. Cicuta, P. Sengupta, A. Honerkamp-Smith, D. Holowka and B. Baird: Critical Fluctuations in Plasma Membrane Vesicles.  ACS Chem. Biol. 3(5): 287-293, 2008.

Holowka, D. and B. Baird: Nanodomains in Early and Later Phases of FceRI Signaling.  Essays in Biochemistry: Membrane Nanodomains, Parmryd, I., ed., Portland Press, London, 57: 147-163, 2015. (featured cover)

Bag, N., D.A. Holowka, B.A. Baird:  Imaging FCS Delineates Subtle Heterogeneity in Plasma Membranes of Resting Mast Cells.  Mol. Biol. Cell 31(7): 709-723, 2020.  (special issue on Quantitative Cell Biology)

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Quantitative fluorescence spectroscopy and microscopy to investigate structural interactions:

Our research group was the first to map the structure of IgE bound to receptors on plasma membrane surfaces, using fluorescence resonance energy transfer. We continue to use quantitative fluorescence spectroscopy and microscopy imaging to investigate changes in structural interactions and distributions that occur in cells that are stimulated by external agents or are pathologically perturbed.

Selected Publications:

Baird, B., Y. Zheng and D. Holowka: Structural mapping of IgE-FceRI, an immuno-receptor complex.  Accts. Chem. Res. 26: 428-434, 1993.

Sengupta, P., D. Holowka and B. Baird: Fluorescence Resonance Energy Transfer between Lipid Probes Detects Nanoscopic Heterogeneity in the Plasma Membrane of Live Cells.  Biophys. J. 92(10): 3564-3574, 2007.

Calloway, N., D. Holowka and B. Baird: A Basic Sequence in STIM1 Promotes Ca2+ Influx by Interacting with the C-Terminal Acidic Coiled-Coil of Orai1.  Biochemistry 49(6): 1067-1071, 2010.

Gadi, D., A. Wagenknecht Wiesner, D. Holowka and B. Baird: Sequestration of Phosphoinositides by Mutated MARCKS Effector Domain Inhibits Stimulated Ca2+ Mobilization and Degranulation in Mast Cells.  Mol. Biol. Cell 22(24): 4908-4917, 2011.

Ramezani, M.,+ M.M. Wilkes,+ T. Das, D. Holowka, D. Eliezer* and B. Baird*:  Regulation of exocytosis and mitochondrial relocalization by Alpha-synuclein in a mammalian cell model.  npj Parkinson’s Disease 5(1): 1-17, 2019.

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Nanoscale-resolution microscopy and micro/nano fabrication to investigate spatio-temporally controlled assemblies:

We further utilize nanoscale-resolution microscopy and micro/nano fabrication, together with biochemical and molecular genetic approaches to investigate how spatio-temporally controlled assemblies of receptors and signaling components.

Selected Publications:

Wu, M., D. Holowka, H.G. Craighead and B. Baird: Visualization of plasma membrane compartmentalization with patterned lipid bilayers. Proc. Natl. Acad. Sci. USA 101(38): 13798-13803, 2004.

Shelby, S.A., D. Holowka, B. Baird and S.L. Veatch:  Distinct Stages of Stimulated FcεRI Receptor Clustering and Immobilization Are Identified through Superresolution Imaging.  Biophys. J. 105(10): 2343-2354, 2013.  (featured covered article: New and Notable)

Singhai, A., D.L. Wakefield, K.L. Bryant, S.R. Hammes, D. Holowka and B. Baird: Spatially defined EFG receptor activation reveals an F-actin-dependent phospho-Erk signaling complex. Biophys. J. 107: 2639-2651, 2014. (featured covered article: New and Notable)

Shelby, S.A., S.L. Veatch, D.A. Holowka and B.A. Baird: Functional nanoscale coupling of Lyn kinase with IgE-FcεRI is restricted by the actin cytoskeleton in early antigen-stimulated signaling. Mol. Biol. Cell 27(22): 3645-3658, 2016. (special issue on Quantitative Cell Biology)

Wakefield, Devin L., D. Holowka and B. Baird: The FceRI Signaling Cascade and Integrin Trafficking Converge at Patterned Ligand Surfaces.  Mol. Biol. Cell 28(23): 3383-3396, 2017. (special issue on Quantitative Cell Biology)

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Fundamental parameters of ligands binding to cell surface receptors:

Using experimental and theoretical approaches, we have characterized fundamental parameters of ligands binding to cell surface receptors, and constructed structurally defined ligands to elucidate features of receptor crosslinking that are critical for hierarchical stages of cell signaling.

Selected Publications:

Erickson, J., B. Goldstein, D. Holowka and B. Baird: The Effect of Receptor Density on the Forward Rate Constant for Binding of Ligands to Cell Surface Receptors.  Biophys. J. 52: 657-662, 1987.

Baird, E.J., D. Holowka, G.W. Coates and B. Baird: Highly Effective Poly(Ethylene Glycol) Architectures for Specific Inhibition of Immune Receptor Activation.  Biochemistry 42(44): 12739-12748, 2003.

Sil, D., J.B. Lee, D. Luo, D. Holowka and B. Baird: Trivalent Ligands with Rigid DNA Spacers Reveal Structural Requirements for IgE Receptor Signaling in RBL Mast Cells.  ACS Chem. Biol. 2(10): 674-684, 2007.

Chylek, L.A., D.A. Holowka, B.A. Baird and W.S. Hlavacek: An interaction library for the FceRI signaling network.  Front. Immunol. 5: 172, 2014.

Mitra, E.D., S.C. Whitehead, D.A. Holowka, B.A. Baird* and J.P. Sethna*: Computation of a Theoretical Membrane Phase Diagram, and the Role of Phase in Lipid Raft-Mediated Protein Organization.  J. Phys. Chem. B 122(13), 3500-3513, 2018.

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Mechanisms by which early assemblies of signaling components link to intracellular response processes:

We characterize the mechanisms by which early assemblies of signaling components link to intracellular response processes, including Ca2+ mobilization, membrane trafficking, and secretory events.

Selected Publications:

Calloway, N., M. Vig, J.-P. Kinet, D. Holowka and B. Baird: Molecular Clustering of STIM1 with Orai1/CRACM1 at the Plasma Membrane Depends Dynamically on Depletion of Ca2+ Stores and on Electrostatic Interactions.  Mol. Biol. Cell 20(1): 389-399, 2009.

Cohen, R., K. Corwith, D. Holowka and B. Baird: Spatiotemporal Resolution of Mast Cell Granule Exocytosis Reveals Correlation with Ca2+ Wave Initiation.  J. Cell Sci. 125(12): 2986-2994, 2012.

Wilkes, M.M., J.D. Wilson, B. Baird and D. Holowka: Activation of Cdc42 is Necessary for Sustained Oscillations of Ca2+ and PIP2 Stimulated by Antigen in RBL Mast Cells.  Biology Open 3(8): 700-710, 2014.

Wilson, J.D., S.A. Shelby, D. Holowka, and B. Baird: Rab11 Regulates the Mast Cell Exocytic Response. Traffic 17(9): 1027-1041, 2016.

Korzeniowski, M.K., E. Wisniewski, B. Baird, D.A. Holowka and T. Balla: Molecular anatomy of the early events in STIM1 activation – oligomerization or conformational change?  J. Cell Sci. 130(17): 2821-2832, 2017.

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Using synthetic materials to interogate cellular structures and processes:

We collaborate with other groups in chemistry, applied physics, and materials science to evaluate impact of new materials on cellular structures and processes.

Selected Publications:

Ow, H., D.R. Larson, M. Srivastava, B.A. Baird, W.W. Webb and U. Wiesner: Bright and Stable Core-Shell Fluorescent Silica Nanoparticles.  Nano Lett. 5(1): 113-117, 2005.

Moran-Mirabal, J.M., A.J. Torres, K.T. Samiee, B.A. Baird and H.G. Craighead: Cell investigation of nanostructures: zero-mode waveguides for plasma membrane studies with single molecule resolution.  Nanotechnology 18(19): 195101-195111, 2007.

Chiang, E.N., R. Dong, C.K. Ober and B.A. Baird: Cellular Responses to Patterned Poly(acrylic acid) Brushes.  Langmuir 27(11): 7016-7023, 2011.

Welch, M. Elizabeth, N.L. Ritzert, H. Chen, N.L. Smith, M.E. Tague, Y. Xu, B.A. Baird, H.D. Abruña and C.K. Ober: Generalized Platform for Antibody Detection using the Antibody Catalyzed Water Oxidation Pathway.  J. Am. Chem. Soc. 136(5): 1879-1883, 2014.

Sun, C., D.L. Wakefield, Y. Han, D.A. Muller, D.A. Holowka, B.A. Baird and W.R. Dichtel: Graphene Oxide Nanosheets Stimulate Ruffling and Shedding of Mammalian Cell Plasma Membranes. Chem 1(2): 273-286, 2016.

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