Anoctamin/TMEM16 Proteins in Lipid Scrambling and Membrane Signaling Open Access
Whitlock, Jarred M. (Fall 2018)
Abstract
The asymmetric organization of membrane lipids is a hallmark of the eukaryotic plasma membrane and is vital for proper cell function. This asymmetry arises from the organization of membrane lipids according to their headgroups via a system of membrane resident lipid transporters and typically results in an extracellular leaflet enriched in phosphatidylcholine and sphingomyelin and a cytosolic leaflet that retains phosphatidylethanolamine and phosphatidylserine. For >35 years it has been recognized that many cells possess the ability to rapidly break down this lipid asymmetry in a Ca2+-dependent manner as a signaling process dubbed phospholipid scrambling. This membrane scrambling exposes cytosolic lipid headgroups (e.g. phosphatidylserine) to the extracellular face, where some are recognized as ligands in cell-cell signaling processes. Recently, members of the anoctamin family of membrane proteins have been recognized for their putative roles in this process. Here I summarize my contribution to the recognition of some anoctamin proteins as phospholipid scramblases, the understanding of how the structure of anoctamins facilitate lipid scrambling, and to the understanding of how anoctamin scramblases regulate biological processes.
Table of Contents
Preface 1
Chapter I Anoctamins: A decade at light-speed
ANO1/TMEM16: From tumor factor to Cl- Channel 6
ANO1 promotes cell proliferation and metastasis
ANO1 regulates embryonic development
ANO1 and ANO2 are bona fide Ca2+-activated Cl- Channels
Most ANOs may elicit Ca2+-dependent phospholipid scrambling, not Cl- conductance 15
Lipid Bilayers: From oily coat, to asymmetric lipid bilayer
Phospholipid Scrambling: a dynamic cell-cell signaling phenomenon Ca2+-dependent phospholipid scrambling: A common membrane signaling phenomenon lacking a molecular identity ANO scramblases
Chapter II Anoctamins/TMEM16 Proteins: Chloride channels flirting with lipids and extracellular vesicles
Most Anoctamins are phospholipid scramblases, not Cl- channels 30
Phospholipid scrambling is a ubiquitous cellular signaling mechanism 33
Phospholipids are organized asymmetrically in the plasma membrane
Externalization of phosphatidylserine and phosphatidylethanolamine to the extracellular leaflet is regulated
Mechanisms of phospholipid scrambling 37
Phospholipid scramblases are a type of channel for lipid head groups
Relationship of ion transport and phospholipid scrambling
Recognition of anionic phospholipids by receptors 42
Externalized anionic phospholipids are recognized by soluble and cell-surface receptors
Exposed anionic phospholipids functions as a signaling platform
Phospholipid scrambling regulates membrane curvature 45
Changes in membrane curvature lead to extracellular vesicle production 47
Extracellular vesicle nomenclature, definition, and biogenesis ANO6 regulates extracellular vesicle release associated with phospholipid scrambling
Extracellular vesicles have two major functions: scaffolding and communication
The role of phospholipid scrambling in membrane fusion 51
Myoblast fusion involves phosphatidylserine exposure
Topic Sidebar: Annexins in phosphatidylserine 52
Exposure
Sperm-egg interaction
Osteoclasts and multinucleated giant cells
Loose Ends: IST2 and endoplasmic reticulum-plasma membrane junctions 58
Chapter III A Pore Idea: The ion conduction pathway of TMEM16/ANO proteins is composed partly of lipid.
The TMEM16 family 65
Phospholipid scrambling
TMEM16 proteins that have diverse functions and many are linked to phospholipid scrambling
Phospholipid scrambling by TMEM16F and homologs
TMEM16A may have evolved from phospholipid scramblases 75
The proteolipidic pore hypothesis 76
TMEM16F conductance is a non-selective leak through the hydrophilic furrow
Fungal afTMEM16 has a large, lipid-dependent, non-selective ion conductance Cl- conduction through TMEM16A occurs via the hydrophilic furrow TMEM16A blockers are hydrophobic and a little weird
The role of lipids in ion channel pores 97
Chapter IV Identification of a lipid scrambling domain in ANO6/TMEM16F
Introduction 104
Results 107
ANO6 expression induces robust phospholipid scrambling in HEK cells
ANO6 current activates in parallel with phospholipid scrambling
ANO6 current and phospholipid scrambling require the same Ca2+ concentration for activation
ANO6 current is non-selective
Identification of a protein domain required for scrambling Ionic currents associated with scrambling Homology model of ANO6
Discussion 132
The scrambling pathway of ANO6
Is ANO6 a phospholipid scramblase
The ion conduction pathway
Evolution of the ANO/TMEM16 family
Is ANO1 a phospholipid scramblase
Materials and Methods 135
Chapter V Muscle Progenitor Cell Fusion In The Maintenance Of Skeletal Muscle
Skeletal muscle requires rapid repair/regeneration mechanisms for lifelong maintenance 151
Plasma membrane lesions undergo patching via Ca2+ regulated exocytic repair
Skeletal muscle employs a multipotent stem cell population in fiber repair/regeneration
Satellite cell dependent muscle repair: A trip back to development? 154
Satellite cells become activated and migrate to tissue damage upon muscle injury
Proliferation of myogenic daughter cells for contribution to the musculature
Satellite cell differentiation
Muscle fusion in fiber repair and regeneration
Subtopic: Muscle by the models 170
Historical use of chick and quail muscle progenitor cells
The use of C. elegans for characterizing myofibril ultrastructure
D. melanogaster in in vivo visualization of myogenesis and muscle fusion
Genetic murine models and the isolation of primary fibers and muscle precursor cells
Zebrafish as a vertebrate in vivo imaging model
Chapter VI Defective membrane fusion and repair in Anoctamin5-deficient muscular dystrophy
Introduction 179
Results 180
Generation of an Ano5-/- mouse model
Clinical and histopathological evaluation of the Ano5-/- mouse
Ano5 facilitates membrane repair
Impaired regeneration in Ano5-/- mice
Loss of Ano5 leads to myoblast fusion defect
Discussion 194
Materials and Methods 197
Chapter VII Anoctamin 5/TMEM16E Ca2+-dependent phospholipid scrambling facilitates muscle precursor cell fusion.
Introduction 216
Results 219
ANO5 elicits phospholipid scrambling
ANO5 phospholipid scrambling is associated with non-selective ionic currents
Muscle progenitor cell fusion and phosphatidylserine exposure is defective in Ano5-/- MPCs
MPC phospholipid scrambling and fusion are rescued by infection with ANO5-virus
Discussion 237
Are phospholipid scrambling-associated currents simply a consequence of PLS or are they biologically significant
Why is Ca2+-dependent phospholipid scrambling defective in Ano5-/- MPCs despite Ano6 expression
Materials and Methods 240
Chapter VIII Conclusion
Overview 248
Summary and significance 248
A look forward 250
Extracellular vesicles as long range signals of Ca2+-dependent phospholipid scrambling
The role of phosphatidylserine exposure during skeletal muscle fusion
The plasma membrane as an oily battery
References 257
Figure Index
Table 1-1: Anoctamins are commonly described using diverse, non-official nomenclature 8
Figure 1-1: ANO PLSases thin membranes and create an energetically feasible pathway for lipid scrambling 24
Figure 2-1: Anoctamins are diverse and cause human disease 32
Figure 2-2: Phospholipids have different shapes that determine membrane curvature which is altered during PLS 35
Figure 2-3: The passive diffusion model of ANO-scramblase function 40
Figure 2-4: PtdSer receptors and the role of PLS in cell-cell signaling 44
Figure 3-1: The TMEM16/Anoctamin (ANO) family tree 68
Figure 3-2: Phospholipid scrambling is a ubiquitous cell signaling process 70
Figure 3-3: Phospholipid scrambling by TMEM16 proteins 74
Figure 3-4: Hypothesis for evolution of a Cl- channel from a phospholipid scramblase 81
Figure 3-5: The TMEM16A furrow likely forms the conduction pathway for Cl- 85
Figure 3-6: Lipid headgroups may form part of the Cl- conductance pathway in TMEM16A 89
Figure 3-7: TMEM16A blockers are hydrophobic molecules 94
Figure 4-1: Expression of ANO6 HEK cells stimulates Ca2+-PLS 110
Figure 4-2: Characteristics of PLS linked to ANO6 113
Figure 4-3: ANO6 current activates coincidently with PLS 116
Figure 4-4: Activation of ANO6 current and PLS requires high intracellular Ca2+ concentrations 118
Figure 4-5: Ionic selectivity of ANO6 currents 120
Figure 4-6: Identification of a PLS domain in ANO6 121
Figure 4-7: Properties of chimeras of ANO1 and ANO6 125
Figure 4-8: Ion channel properties of ANO1-ANO6 chimeras 128
Figure 4-9: Homology model of ANO6 131
Figure 4-S6-1: Genebank accession numbers of sequences of mammalian species ANO1 and ANO6 used for DIVERGE analysis 142
Figure 4-S6-2: MUSCLE alignment of mANO1(ac) and mANO6 used for constructing chimeras 143
Figure 4-S6-3: Properties of 1-6-1 chimeras that trafficked to the plasma membrane and generated ionic currents 144
Figure 4-S6-4: Properties of 1-6-1 chimeras in which pairs or triples of amino acids were mutated 145
Figure 4-S6-5: Properties of ANO6 with mutations in the SCRD 145
Figure 4-S7-1: Patch clamp analysis of ANO1-ANO6 chimeras 146
Figure 4-S9-1: Homology model of ANO1 dimer 147
Figure 5-1: An illustrative representation of skeletal muscle cell ultrastructure 151
Figure 5-2: Satellite cell-dependent skeletal muscle repair 159
Figure 5-3: Myogenic progression in satellite cell-dependent 162
Figure 6-1: Generation of the Ano5-/- mouse model 182
Figure 6-2: Characterization of Ano5-/- deficient mice 184
Figure 6-3: Subcellular histopathology in Ano5-/- muscle 187
Figure 6-4: Membrane repair is defective in Ano5-/- mice 191
Figure 6-5: Loss of Ano5 expression impairs myoblast fusion 194
Figure 6-S1: ANO5 mutations associated with myopathy 209
Figure 6-S2: Relative expression of Ano6 in Ano5-/- muscles 210
Figure 6-S3: Physiological characterization of Ano5-/- mice 211
Figure 6-S4: Ano5-/- mouse histologically phenocopies LGMD2L Patient 212
Figure 6-S5: Loss of Ano5-/- Alters citrate synthase activity 213
Figure 7-1: ANO5 expression activates Ca2+-PLS 222
Figure 7-2: ANO5-dependent Ca2+-PLS is associated with an ionic current 225
Figure 7-3: ANO5-PLS associated ionic currents are non-selective 226
Figure 7-4: Exogenous “gene trap” knock-in results in loss of ANO5 229
Figure 7-5: Ano5-/- muscle cells exhibit perturbed Ca2+-PLS and PLS-associated ionic current. 231
Figure 7-6: Exogenous ANO5 expression rescues Ano5-/- MPC fusion 234
Figure 7-7: Exogenous ANO5 expression rescues Ano5-/- MPC Ca2+-PLS 236
Figure 7-S1: ANO5 expression rescues Ano5-/- MPC fusion 237
Table 8-1: The major lipid species of the plasma membrane exhibit asymmetrical sidedness 255
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