The plasma membrane (PM) forms the physical barrier and functional interface between a cell and its environment. To accommodate this complexity, the functionality of the PM is amplified by compartmentalization into compositionally and functionally distinct lateral domains, of which lipid rafts are the archetypal example. Raft-mediated signal transduction has been extensively implicated in diverse cell functions, with dysregulation contributing to the aberrant signaling in cancer, hyperinflammation, autoimmunity, and cardiovascular disease. Despite this potential impact, a dearth of consistent, quantitative methodologies has prevented clear definition of raft composition or unequivocal mechanistic description of raft function. A recent methodological breakthrough is the direct observation of large-scale ordered domains in plasma membranes isolated from mammalian cells. This system confirms the inherent capacity of mammalian PMs to form raft domains and also provides a robust experimental platform for direct, quantitative investigations into their composition and physical properties. We propose a comprehensive approach combining biophysics, bioinformatics, in silico molecular modeling, and cell biology to characterize the structural determinants and functional consequences of protein partitioning to PM microdomains. Our extensive preliminary data reveal that protein transmembrane domains (TMDs) encompass the necessary determinants for raft affinity.
In Aim 1, we will define the general TMD physical features that impart raft affinity, focusing specifically on TMD length and surface area to test the hypothesis that relatively long and thin TMDs have more favorable interactions with ordered membrane microenvironments. Experimental measurements of raft partitioning will be supported by computational modeling and bioinformatics with the ultimate goal of generating a physical model that can identify raft preferring proteins from amino acid sequence.
In Aim 2, we will extend the study from single TMDs to evaluate the role of TMD oligomerization in driving raft affinity. Our preliminary data has identified a specific TMD sequence motif that significantly enhances raft phase association. We will evaluate the hypothesis that such enhancement is driven by TMD oligomerization via quantitative evaluation of TMD oligomerization and its effect on raft partitioning in live cells, isolated PMs, and synthetic model systems. The structural details behind these observations will be investigated by atomistic molecular modeling. Finally, we aim to definitively demonstrate raft affinity as a major regulator of subcellular membrane traffic by the experiments proposed in Aim 3. To this end, we have generated a panel of protein variants lacking any sorting determinants except their TMD-encoded raft affinity. For these proteins, PM recycling after endocytosis relies on their partitioning into ordered membrane domains, implying a raft- mediated protein sorting mechanism. The trafficking pathways and molecular machinery underlying this mechanism will be investigated by imaging experiments using the TMD panel as validated probes of raft and non-raft domains. These studies will identify proteins that rely on microdomain association for their function, define the physicochemical nature of this association, and clarify the mechanisms by which PM organization regulates cell physiology. The long-term goal is to facilitate rational design of small molecules that interfere with protein association with microdomains in disease states defined by aberrant PM signal transduction.

Public Health Relevance

(Public Health Relevance) Accurate signal transduction and transmission is an absolute prerequisite for optimal cell function, with aberrations in cell signaling responsible for the majority of diseases. Most signals are received by cells at the plasma membrane, which forms the physical barrier and functional interface between a cell and its environment. To ensure accurate signal transmission, membranes are subdivided into functional compartments, or domains, that segregate membrane proteins and lipids for optimal functionality. Although there is clear evidence that plasma membranes are compartmentalized, and that disruption of this compartmentalization leads to dysfunction, it remains unclear how proteins are recruited to specific domains and how this recruitment affects cell function. We are investigating these basic questions with the long-term goal of designing drugs that interfere with membrane partitioning for treatment of disease states defined by aberrant plasma membrane signal transduction, including cancer, autoimmune disease, and cardiovascular disease. !

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM124072-02S1
Application #
9733413
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Preusch, Peter
Project Start
2017-09-30
Project End
2021-08-31
Budget Start
2018-09-01
Budget End
2019-08-31
Support Year
2
Fiscal Year
2018
Total Cost
Indirect Cost
Name
University of Texas Health Science Center Houston
Department
Biology
Type
Schools of Medicine
DUNS #
800771594
City
Houston
State
TX
Country
United States
Zip Code
77030
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Cornell, Caitlin E; Skinkle, Allison D; He, Shushan et al. (2018) Tuning Length Scales of Small Domains in Cell-Derived Membranes and Synthetic Model Membranes. Biophys J 115:690-701
Ernst, Robert; Ballweg, Stephanie; Levental, Ilya (2018) Cellular mechanisms of physicochemical membrane homeostasis. Curr Opin Cell Biol 53:44-51
Levental, Kandice R; Surma, Michal A; Skinkle, Allison D et al. (2017) ?-3 polyunsaturated fatty acids direct differentiation of the membrane phenotype in mesenchymal stem cells to potentiate osteogenesis. Sci Adv 3:eaao1193
Lorent, Joseph H; Diaz-Rohrer, Blanca; Lin, Xubo et al. (2017) Structural determinants and functional consequences of protein affinity for membrane rafts. Nat Commun 8:1219