All organisms, from single-celled bacteria to multi-cellular animals and plants, must sense and respond to mechanical force in their external environment (shear force, gravity, touch) and in their internal environment (osmotic pressure, membrane deformation) for proper growth, development, and health. Our research focuses on two families of mechanosensitive channels, the prokaryotic channels MscL and MscS and their eukaryotic homologs in Arabidopsis. MscL and MscS are intrinsically stretch-activated channels that open and close in response to tension applied directly to the bilayer and consequently are sensitive reporters of protein- membrane energetics. Elucidating how these mechanosensitive channels function in the context of the membrane will help us understand how mechanical force can generate biophysical alterations that in turn lead to adaptive changes in cell physiology.
Aim 1 : Investigate the crystal structures of MscL and MscS in multiple conformational states. MscL and MscS are among the few gated channels that have been crystallographically determined. Our highest priority is to improve the structures of the E. coli channels, but we will also systematically survey prokaryotic homologs and the use of molecular doorstops to trap channels in alternate conformational states to define the gating transition in structural detail.
Aim 2 : Analyze the biophysical interactions between mechanosensitive channels and the lipid bilayer. Working within the context of a theoretical model, the coupling between gating tension, bilayer thickness, and width of the hydrophobic region of MscL will be explored through mutagenesis and single channel electrophysiology. These studies will dissect the energetic contributions of different membrane deformation terms to the conformational equilibrium between channel states. The physiologically crucial permeation of water through MscL and MscS in giant unilamellar vesicles will be measured volumetrically and compared to the fluid transport properties anticipated from conductance measurements.
Aim 3 : Characterize functional and structural aspects of eukaryotic MscS-Like channels. The MscS-Like (MSL) channels of Arabidopsis provide an opportunity to investigate the structure and function of mechanosensitive channels in the context of multi-cellular eukaryotic organisms. The oligomeric state, channel characteristics, and structure of these proteins will be investigated. We will also use a series of new and established assays to characterize their biological function in osmotic shock protection, intramembrane localization, electrophysiology and organelle morphology control. Our proposed experiments on the MSLs, together with the experiments proposed above for MscL and MscS, are the start towards a systematic approach to revealing how MS channels function in the context of the membrane and the cell.

Public Health Relevance

Force-sensing is a critical aspect of healthy cell growth, morphology and development. We will study in molecular detail how force-sensing is achieved by two families of stretch-activated membrane channels.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM084211-02
Application #
7684651
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Chin, Jean
Project Start
2008-09-15
Project End
2012-08-31
Budget Start
2009-09-01
Budget End
2010-08-31
Support Year
2
Fiscal Year
2009
Total Cost
$376,147
Indirect Cost
Name
California Institute of Technology
Department
Chemistry
Type
Schools of Engineering
DUNS #
009584210
City
Pasadena
State
CA
Country
United States
Zip Code
91125
Maksaev, Grigory; Shoots, Jennette M; Ohri, Simran et al. (2018) Nonpolar residues in the presumptive pore-lining helix of mechanosensitive channel MSL10 influence channel behavior and establish a nonconducting function. Plant Direct 2:
Herrera, Nadia; Maksaev, Grigory; Haswell, Elizabeth S et al. (2018) Elucidating a role for the cytoplasmic domain in the Mycobacterium tuberculosis mechanosensitive channel of large conductance. Sci Rep 8:14566
Chure, Griffin; Lee, Heun Jin; Rasmussen, Akiko et al. (2018) Connecting the dots between mechanosensitive channel abundance, osmotic shock, and survival at single-cell resolution. J Bacteriol :
Hamant, Olivier; Haswell, Elizabeth S (2017) Life behind the wall: sensing mechanical cues in plants. BMC Biol 15:59
Jensen, Gregory S; Fal, Kateryna; Hamant, Olivier et al. (2017) The RNA Polymerase-Associated Factor 1 Complex Is Required for Plant Touch Responses. J Exp Bot 68:499-511
Basu, Debarati; Haswell, Elizabeth S (2017) Plant mechanosensitive ion channels: an ocean of possibilities. Curr Opin Plant Biol 40:43-48
Einav, Tal; Phillips, Rob (2017) Monod-Wyman-Changeux Analysis of Ligand-Gated Ion Channel Mutants. J Phys Chem B 121:3813-3824
Lee, Chun Pong; Maksaev, Grigory; Jensen, Gregory S et al. (2016) MSL1 is a mechanosensitive ion channel that dissipates mitochondrial membrane potential and maintains redox homeostasis in mitochondria during abiotic stress. Plant J 88:809-825
Walton, Troy A; Idigo, Chinenye A; Herrera, Nadia et al. (2015) MscL: channeling membrane tension. Pflugers Arch 467:15-25
Hamilton, Eric S; Schlegel, Angela M; Haswell, Elizabeth S (2015) United in diversity: mechanosensitive ion channels in plants. Annu Rev Plant Biol 66:113-37

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