Anoctamin/TMEM16 Proteins in Lipid Scrambling and Membrane Signaling Open Access

Whitlock, Jarred M. (Fall 2018)

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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


           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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