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      ANO5 ensures trafficking of annexins in wounded myofibers

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          Abstract

          Mutations in ANO5 cause a limb-girdle muscular dystrophy characterized by membrane repair defects that make muscles more susceptible to permanent damage from normal use. This work links failed membrane repair in ANO5-deficient muscle to altered trafficking of the annexins, crucial repair proteins.

          Abstract

          Mutations in ANO5 ( TMEM16E) cause limb-girdle muscular dystrophy R12. Defective plasma membrane repair is a likely mechanism. Using myofibers from Ano5 knockout mice, we show that trafficking of several annexin proteins, which together form a cap at the site of injury, is altered upon loss of ANO5. Annexin A2 accumulates at the wound to nearly twice the level observed in WT fibers, while annexin A6 accumulation is substantially inhibited in the absence of ANO5. Appearance of annexins A1 and A5 at the cap is likewise diminished in the Ano5 knockout. These changes are correlated with an alteration in annexin repair cap fine structure and shedding of annexin-positive vesicles. We conclude that loss of annexin coordination during repair is disrupted in Ano5 knockout mice and underlies the defective repair phenotype. Although ANO5 is a phospholipid scramblase, abnormal repair is rescued by overexpression of a scramblase-defective ANO5 mutant, suggesting a novel, scramblase-independent role of ANO5 in repair.

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          Analyzing real-time PCR data by the comparative CT method

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            Analyzing real-time PCR data by the comparative C(T) method.

            Two different methods of presenting quantitative gene expression exist: absolute and relative quantification. Absolute quantification calculates the copy number of the gene usually by relating the PCR signal to a standard curve. Relative gene expression presents the data of the gene of interest relative to some calibrator or internal control gene. A widely used method to present relative gene expression is the comparative C(T) method also referred to as the 2 (-DeltaDeltaC(T)) method. This protocol provides an overview of the comparative C(T) method for quantitative gene expression studies. Also presented here are various examples to present quantitative gene expression data using this method.
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              Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells.

              Many proteins associated with the plasma membrane are known to partition into submicroscopic sphingolipid- and cholesterol-rich domains called lipid rafts, but the determinants dictating this segregation of proteins in the membrane are poorly understood. We suppressed the tendency of Aequorea fluorescent proteins to dimerize and targeted these variants to the plasma membrane using several different types of lipid anchors. Fluorescence resonance energy transfer measurements in living cells revealed that acyl but not prenyl modifications promote clustering in lipid rafts. Thus the nature of the lipid anchor on a protein is sufficient to determine submicroscopic localization within the plasma membrane.
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                Author and article information

                Journal
                J Cell Biol
                J Cell Biol
                jcb
                The Journal of Cell Biology
                Rockefeller University Press
                0021-9525
                1540-8140
                01 March 2021
                26 January 2021
                : 220
                : 3
                : e202007059
                Affiliations
                [1]Department of Cell Biology, Emory University School of Medicine, Atlanta, GA
                Author notes
                Correspondence toH. Criss Hartzell: criss.hartzell@ 123456emory.edu
                Author information
                https://orcid.org/0000-0002-9851-3693
                https://orcid.org/0000-0002-3393-1528
                Article
                jcb.202007059
                10.1083/jcb.202007059
                7844426
                33496727
                1bda33fc-a825-4ac6-ab9c-7847a6291093
                © 2021 Foltz et al.

                This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms/). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/).

                History
                : 14 July 2020
                : 20 November 2020
                : 23 December 2020
                Page count
                Pages: 19
                Funding
                Funded by: Emory University, DOI http://dx.doi.org/10.13039/100006939;
                Funded by: National Institutes of Health, DOI http://dx.doi.org/10.13039/100000002;
                Funded by: National Institute of Arthritis and Musculoskeletal and Skin Diseases, DOI http://dx.doi.org/10.13039/100000069;
                Award ID: R01AR067786
                Award ID: R01AR071397
                Award ID: F32AR074249
                Funded by: National Eye Institute, DOI http://dx.doi.org/10.13039/100000053;
                Award ID: R01EY114852
                Funded by: National Institute of General Medical Sciences, DOI http://dx.doi.org/10.13039/100000057;
                Award ID: R01GM132598
                Categories
                Article
                Physiology
                Trafficking
                Membrane and Lipid Biology
                Disease

                Cell biology
                Cell biology

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