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To obtain accurate, reproducible, and interpretable data when conducting imaging experiments, it is critical to consider external factors affecting data acquisition at various steps of the experimental workflow. Illumination power and stability represent two critical factors, especially when comparing fluorescence intensities between images during a time-lapse experiment or experiments performed at different times or on other microscopes. The fluorescence signal can be generated by different types of light sources. These light sources and their coupling elements (e.g., fibers) can display varying performances over time as they age, move, or as environmental conditions change. Unfortunately, microscope users can often only set illumination power as a percentage of its maximal output and, may therefore, not be aware of potential performance changes. It is important to recognize that a set percentage will not always yield the same illumination power in Watts at the objective over the course of an experiment, not to mention between days or systems. This means that selecting for example 10% output may lead to different experimental results over time and will not necessarily be comparable to outputs obtained from other lasers or microscopes, even those of the very same model. If you are responsible for system maintenance, routinely measuring the illumination power, stability, and linearity over time can help you detect issues that affect the integrity of the system and thus the reproducibility of an experiment. This protocol describes how to measure the stability and linearity of the illumination power using calibrated external power sensors. This protocol is at the moment intended for confocal systems (raster scanning and spinning disks), but will be extended later to other imaging modalities. It represents the collective experience of 60 imaging scientists. Measurements made by our working group with this protocol are available in a public database, which will be updated with further contributions from the community.

Live imaging of neuromuscular junctions (NMJs) in situ has been constrained by the suitability of ligands for inert vital staining of motor nerve terminals. Here, we constructed several truncated derivatives of the tetanus toxin C-fragment (TetC) fused with Emerald Fluorescent Protein (emGFP). Four constructs, namely full length emGFP-TetC (emGFP-865:TetC) or truncations comprising amino acids 1066–1315 (emGFP-1066:TetC), 1093–1315 (emGFP-1093:TetC) and 1109–1315 (emGFP-1109:TetC), produced selective, high-contrast staining of motor nerve terminals in rodent or human muscle explants. Isometric tension and intracellular recordings of endplate potentials from mouse muscles indicated that neither full-length nor truncated emGFP-TetC constructs significantly impaired NMJ function or transmission. Motor nerve terminals stained with emGFP-TetC constructs were readily visualised in situ or in isolated preparations using fibre-optic confocal endomicroscopy (CEM). emGFP-TetC derivatives and CEM also visualised regenerated NMJs. Dual-waveband CEM imaging of preparations co-stained with fluorescent emGFP-TetC constructs and Alexa647-α-bungarotoxin resolved innervated from denervated NMJs in axotomized WldS mouse muscle and degenerating NMJs in transgenic SOD1G93A mouse muscle. Our findings highlight the region of the TetC fragment required for selective binding and visualisation of motor nerve terminals and show that fluorescent derivatives of TetC are suitable for in situ morphological and physiological characterisation of healthy, injured and diseased NMJs.

Most mitochondrial diseases are disabling and progressive, usually affecting multiple organs with high energy demand such as the brain, liver, skeletal muscle and heart. Although defective oxidative phosphorylation is the common final pathway, it is unknown why different mutations result in largely heterogeneous clinical presentations. Currently there are very few effective therapies and treatment is usually symptomatic.

Reversible infantile respiratory chain deficiency (RIRCD) is a rare mitochondrial myopathy leading to severe metabolic disturbances in infants, which recover spontaneously after 6‐months of age. RIRCD is associated with the homoplasmic m.14674T>C mitochondrial DNA mutation; however, only ~ 1/100 carriers develop the disease. We studied 27 affected and 15 unaffected individuals from 19 families and found additional heterozygous mutations in nuclear genes interacting with mt‐tRNAGlu including EARS2 and TRMU in the majority of affected individuals, but not in healthy carriers of m.14674T>C, supporting a digenic inheritance. Our transcriptomic and proteomic analysis of patient muscle suggests a stepwise mechanism where first, the integrated stress response associated with increased FGF21 and GDF15 expression enhances the metabolism modulated by serine biosynthesis, one carbon metabolism, TCA lipid oxidation and amino acid availability, while in the second step mTOR activation leads to increased mitochondrial biogenesis. Our data suggest that the spontaneous recovery in infants with digenic mutations may be modulated by the above described changes. Similar mechanisms may explain the variable penetrance and tissue specificity of other mtDNA mutations and highlight the potential role of amino acids in improving mitochondrial disease., Heterozygous mutations in nuclear genes interacting with mt‐tRNAGlu induce the integrated stress response and metabolic rearrangements, reducing penetrance and promoting spontaneous recovery in a rare mitochondrial myopathy.

Reversible infantile respiratory chain deficiency (RIRCD) is a rare mitochondrial myopathy leading to severe metabolic disturbances in infants, which recover spontaneously after 6 months of age. RIRCD is associated with the homoplasmic m.14674T>C mitochondrial DNA mutation, however only ∼1/100 carriers develop the disease. We studied 27 affected and 15 unaffected individuals from 19 families and found additional heterozygous mutations in nuclear genes interacting with mt-tRNAGluincludingEARS2andTRMUin the majority of affected individuals, but not in healthy carriers of m.14674T>C, supporting a digenic inheritance. The spontaneous recovery in infants with digenic mutations is modulated by changes in amino acid availability in a multi-step process. First, the integrated stress-response associated with increasedFGF21andGDF15expression enhances catabolism via β-oxidation and the TCA cycle increasing the availability of amino acids. In the second phase mitochondrial biogenesis increases via mTOR activation, leading to improved mitochondrial translation and recovery. Similar mechanisms may explain the variable penetrance and tissue specificity of other mtDNA mutations and highlight the potential role of amino acids in improving mitochondrial disease.

Reversible infantile respiratory chain deficiency is characterised by spontaneous recovery of mitochondrial myopathy in infants. We studied whether a physiological isoform switch of nuclear cytochrome c oxidase subunits contributes to the age-dependent manifestation and spontaneous recovery in reversible mitochondrial disease. Some nuclear-encoded subunits of cytochrome c oxidase are present as tissue-specific isoforms. Isoforms of subunits COX6A and COX7A expressed in heart and skeletal muscle are different from isoforms expressed in the liver, kidney and brain. Furthermore, in skeletal muscle both the heart and liver isoforms of subunit COX7A have been demonstrated with variable levels, indicating that the tissue-specific expression of nuclear-encoded subunits could provide a basis for the fine-tuning of cytochrome c oxidase activity to the specific metabolic needs of the different tissues. We demonstrate a developmental isoform switch of COX6A and COX7A subunits in human and mouse skeletal muscle. While the liver type isoforms are more present soon after birth, the heart/muscle isoforms gradually increase around 3 months of age in infants, 4 weeks of age in mice, and these isoforms persist in muscle throughout life. Our data in follow-up biopsies of patients with reversible infantile respiratory chain deficiency indicate that the physiological isoform switch does not contribute to the clinical manifestation and to the spontaneous recovery of this disease. However, understanding developmental changes of the different cytochrome c oxidase isoforms may have implications for other mitochondrial diseases. This article is part of a Directed Issue entitled: Energy Metabolism Disorders and Therapies.

Mitochondrial dysfunction and altered proteostasis are central features of neurodegenerative diseases. The pitrilysin metallopeptidase 1 (PITRM1) is a mitochondrial matrix enzyme, which digests oligopeptides, including the mitochondrial targeting sequences that are cleaved from proteins imported across the inner mitochondrial membrane and the mitochondrial fraction of amyloid beta (Aβ). We identified two siblings carrying a homozygous PITRM1 missense mutation (c.548G>A, p.Arg183Gln) associated with an autosomal recessive, slowly progressive syndrome characterised by mental retardation, spinocerebellar ataxia, cognitive decline and psychosis. The pathogenicity of the mutation was tested in vitro, in mutant fibroblasts and skeletal muscle, and in a yeast model. A Pitrm1 +/− heterozygous mouse showed progressive ataxia associated with brain degenerative lesions, including accumulation of Aβ‐positive amyloid deposits. Our results show that PITRM1 is responsible for significant Aβ degradation and that impairment of its activity results in Aβ accumulation, thus providing a mechanistic demonstration of the mitochondrial involvement in amyloidotic neurodegeneration.

Objective:To study the prevalence, molecular cause, and clinical presentation of hereditary motor neuropathies in a large cohort of patients from the North of England.Methods:Detailed neurologic and electrophysiologic assessments and next-generation panel testing or whole exome sequencing were performed in 105 patients with clinical symptoms of distal hereditary motor neuropathy (dHMN, 64 patients), axonal motor neuropathy (motor Charcot-Marie-Tooth disease [CMT2], 16 patients), or complex neurologic disease predominantly affecting the motor nerves (hereditary motor neuropathy plus, 25 patients).Results:The prevalence of dHMN is 2.14 affected individuals per 100,000 inhabitants (95% confidence interval 1.62–2.66) in the North of England. Causative mutations were identified in 26 out of 73 index patients (35.6%). The diagnostic rate in the dHMN subgroup was 32.5%, which is higher than previously reported (20%). We detected a significant defect of neuromuscular transmission in 7 cases and identified potentially causative mutations in 4 patients with multifocal demyelinating motor neuropathy.Conclusions:Many of the genes were shared between dHMN and motor CMT2, indicating identical disease mechanisms; therefore, we suggest changing the classification and including dHMN also as a subcategory of Charcot-Marie-Tooth disease. Abnormal neuromuscular transmission in some genetic forms provides a treatable target to develop therapies.

AIMS:The organization and maturation of ventricular cardiomyocytes from the embryonic to the adult form is crucial for normal cardiac function. We have shown that a polarity protein, Scrib, may be involved in regulating the early stages of this process. Our goal was to establish whether Scrib plays a cell autonomous role in the ventricular myocardium, and whether this involves well-known polarity pathways.METHODS AND RESULTS:Deletion of Scrib in cardiac precursors utilizing Scrib(flox) mice together with the Nkx2.5-Cre driver resulted in disruption of the cytoarchitecture of the forming trabeculae and ventricular septal defects. Although the majority of mice lacking Scrib in the myocardium survived to adulthood, they developed marked cardiac fibrosis. Scrib did not physically interact with the planar cell polarity (PCP) protein, Vangl2, in early cardiomyocytes as it does in other tissues, suggesting that the anomalies did not result from disruption of PCP signalling. However, Scrib interacted with Rac1 physically in embryonic cardiomyocytes and genetically to result in ventricular abnormalities, suggesting that this interaction is crucial for the development of the early myocardium.CONCLUSIONS:The Scrib-Rac1 interaction plays a crucial role in the organization of developing cardiomyocytes and formation of the ventricular myocardium. Thus, we have identified a novel signalling pathway in the early, functioning, heart muscle. These data also show that the foetus can recover from relatively severe abnormalities in prenatal ventricular development, although cardiac fibrosis can be a long-term consequence.

Background Mitochondrial encephalomyopathies are severe, relentlessly progressive conditions and there are very few effective therapies available to date. We have previously suggested that in two rare forms of reversible mitochondrial disease (reversible infantile respiratory chain deficiency and reversible infantile hepatopathy) supplementation with L-cysteine can improve mitochondrial protein synthesis, since cysteine is required for the 2-thiomodification of mitochondrial tRNAs. Objectives We studied whether supplementation with L-cysteine or N-acetyl-cysteine (NAC) results in any improvement of the mitochondrial function in vitro in fibroblasts of patients with different genetic forms of abnormal mitochondrial translation. Methods We studied in vitro in fibroblasts of patients carrying the common m.3243A>G and m.8344A>G mutations or autosomal recessive mutations in genes affecting mitochondrial translation, whether L-cysteine or N-acetyl-cysteine supplementation have an effect on mitochondrial respiratory chain function. Results Here we show that supplementation with L-cysteine, but not with N-acetyl-cysteine partially rescues the mitochondrial translation defect in vitro in fibroblasts of patients carrying the m.3243A>G and m.8344A>G mutations. In contrast, N-acetyl-cysteine had a beneficial effect on mitochondrial translation in TRMU and MTO1 deficient fibroblasts. Conclusions Our results suggest that L-cysteine or N-acetyl-cysteine supplementation may be a potential treatment for selected subgroups of patients with mitochondrial translation deficiencies. Further studies are needed to explore the full potential of cysteine supplementation as a treatment for patients with mitochondrial disease.

Envoplakin and periplakin are the two smallest plakin family cytoskeletal linker proteins that connect intermediate filaments to cellular junctions and other membrane locations. These two plakins have a structural role in the assembly of the cornified envelope (CE), the terminal stage of epidermal differentiation. Analysis of gene-targeted mice lacking both these plakins and the third initial CE scaffold protein, involucrin, demonstrate the importance of the structural integrity of CE for a proper epidermal barrier function. It has emerged that periplakin, which also has a wider tissue distribution than envoplakin, has additional, independent roles. Periplakin participates in the cytoskeletal organization also in other tissues and interacts with a wide range of membrane-associated proteins such as kazrin and butyrophilin BTN3A1. This review covers methods used to understand periplakin and envoplakin functions in cell culture models, including siRNA ablation of periplakin expression and the use of tagged protein domain constructs to study localization and interactions. In addition, assays that can be used to analyze CEs and epidermal barrier function in gene-targeted mice are described and discussed.

Envoplakin and periplakin are the two smallest plakin family cytoskeletal linker proteins that connect intermediate filaments to cellular junctions and other membrane locations. These two plakins have a structural role in the assembly of the cornified envelope (CE), the terminal stage of epidermal differentiation. Analysis of gene-targeted mice lacking both these plakins and the third initial CE scaffold protein, involucrin, demonstrate the importance of the structural integrity of CE for a proper epidermal barrier function. It has emerged that periplakin, which also has a wider tissue distribution than envoplakin, has additional, independent roles. Periplakin participates in the cytoskeletal organization also in other tissues and interacts with a wide range of membrane-associated proteins such as kazrin and butyrophilin BTN3A1. This review covers methods used to understand periplakin and envoplakin functions in cell culture models, including siRNA ablation of periplakin expression and the use of tagged protein domain constructs to study localization and interactions. In addition, assays that can be used to analyze CEs and epidermal barrier function in gene-targeted mice are described and discussed.

Liver failure is a heterogeneous condition which may be fatal and the primary cause is frequently unknown. We investigated mitochondrial oxidative phosphorylation in patients undergoing liver transplantation. We studied 45 patients who had liver transplantation due to a variety of clinical presentations. Blue native polyacrylamide gel electrophoresis with immunodetection of respiratory chain complexes I-V, biochemical activity of respiratory chain complexes II and IV and quantification of mitochondrial DNA (mtDNA) copy number were investigated in liver tissue collected from the explanted liver during transplantation. Abnormal mitochondrial function was frequently present in this cohort: ten of 40 patients (25 %) had a defect of one or more respiratory chain enzyme complexes on blue native gels, 20 patients (44 %) had low activity of complex II and/or IV and ten (22 %) had a reduced mtDNA copy number. Combined respiratory chain deficiency and reduced numbers of mitochondria were detected in all three patients with acute liver failure. Low complex IV activity in biliary atresia and complex II defects in cirrhosis were common findings. All six patients diagnosed with liver tumours showed variable alterations in mitochondrial function, probably due to the heterogeneity of the presenting tumour. In conclusion, mitochondrial dysfunction is common in severe liver failure in non-mitochondrial conditions. Therefore, in contrast to the common practice detection of respiratory chain abnormalities in liver should not restrict the inclusion of patients for liver transplantation. Furthermore, improving mitochondrial function may be targeted as part of a complex therapy approach in different forms of liver diseases. Electronic supplementary material The online version of this article (doi:10.1007/s10545-016-9927-z) contains supplementary material, which is available to authorized users.

Periplakin is a cytoskeletal linker protein that participates in the assembly of epidermal cell cornified envelope and regulates keratin organisation in simple epithelial cells. We have generated a stably transfected MCF-7 subclone expressing HA-tagged periplakin N-terminus to identify molecular interactions of periplakin. Co-immunoprecipitation with anti-HA antibodies and mass spectrometry identified a 500-kDa periplakin-interacting protein as plectin, another plakin family member. Plectin-periplakin interaction was confirmed by immunoblotting of complexes immunoprecipitated by either anti-HA or anti-plectin antibodies. Transient transfections of periplakin deletion constructs indicated that first 133 amino acid residues of the N-terminus are sufficient for co-localisation with plectin at MCF-7 cell borders. Immunofluorescence analysis demonstrated that periplakin and plectin isoforms 1, 1f and 1k co-localise at cell borders of MCF-7 epithelia and that plectin-1f and 1k co-localise with periplakin in suprabasal epidermis. Ablation of plectin by siRNA in HaCaT keratinocytes resulted in aggregation of periplakin to small clusters. Scratch-wounded MCF-7 epithelia expressing periplakin N-terminus showed accelerated keratin re-organisation that was inhibited by siRNA knock-down of plectin. Finally, ablation of either periplakin or plectin, or both proteins simultaneously, impaired migration of MCF-7 epithelial sheets. Thus, we have identified a novel functional co-localisation between two plakin cytolinker proteins.

Mitochondrial diseases are usually severe and progressive conditions; however, there are rare forms that show remarkable spontaneous recoveries. Two homoplasmic mitochondrial tRNA mutations (m.14674TC/G in mt-tRNA(Glu)) have been reported to cause severe infantile mitochondrial myopathy in the first months of life. If these patients survive the first year of life by extensive life-sustaining measures they usually recover and develop normally. Another mitochondrial disease due to deficiency of the 5-methylaminomethyl-2-thiouridylate methyltransferase (TRMU) causes severe liver failure in infancy, but similar to the reversible mitochondrial myopathy, within the first year of life these infants may also recover completely. Partial recovery has been noted in some other rare forms of mitochondrial disease due to deficiency of mitochondrial tRNA synthetases and mitochondrial tRNA modifying enzymes. Here we summarize the clinical presentation of these unique reversible mitochondrial diseases and discuss potential molecular mechanisms behind the reversibility. Understanding these mechanisms may provide the key to treatments of potential broader relevance in mitochondrial disease, where for the majority of the patients no effective treatment is currently available.

Synaptotagmin 2 is a synaptic vesicle protein that functions as a calcium sensor for neurotransmission but has not been previously associated with human disease. Via whole-exome sequencing, we identified heterozygous missense mutations in the C2B calcium-binding domain of the gene encoding Synaptotagmin 2 in two multigenerational families presenting with peripheral motor neuron syndromes. An essential calcium-binding aspartate residue, Asp307Ala, was disrupted by a c.920A>C change in one family that presented with an autosomal-dominant presynaptic neuromuscular junction disorder resembling Lambert-Eaton myasthenic syndrome. A c.923C>T variant affecting an adjacent residue (p.Pro308Leu) produced a presynaptic neuromuscular junction defect and a dominant hereditary motor neuropathy in a second family. Characterization of the mutation homologous to the human c.920A>C variant in Drosophila Synaptotagmin revealed a dominant disruption of synaptic vesicle exocytosis using this transgenic model. These findings indicate that Synaptotagmin 2 regulates neurotransmitter release at human peripheral motor nerve terminals. In addition, mutations in the Synaptotagmin 2 C2B domain represent an important cause of presynaptic congenital myasthenic syndromes and link them with hereditary motor axonopathies., National Institutes of Health (U.S.) (NIH Grant NS40296), Picower Institute for Learning and Memory (Picower Neurological Disease Research Fund), JPB Foundation

Background Behr’s syndrome is a classical phenotypic description of childhood-onset optic atrophy combined with various neurological symptoms, including ophthalmoparesis, nystagmus, spastic paraparesis, ataxia, peripheral neuropathy and learning difficulties. Objective Here we describe 4 patients with the classical Behr’s syndrome phenotype from 3 unrelated families who carry homozygous nonsense mutations in the C12orf65 gene encoding a protein involved in mitochondrial translation. Methods Whole exome sequencing was performed in genomic DNA and oxygen consumption was measured in patient cell lines. Results We detected 2 different homozygous C12orf65 nonsense mutations in 4 patients with a homogeneous clinical presentation matching the historical description of Behr’s syndrome. The first symptom in all patients was childhood-onset optic atrophy, followed by spastic paraparesis, distal weakness, motor neuropathy and ophthalmoparesis. Conclusions We think that C12orf65 mutations are more frequent than previously suggested and screening of this gene should be considered not only in patients with mitochondrial respiratory chain deficiencies, but also in inherited peripheral neuropathies, spastic paraplegias and ataxias, especially with pre-existing optic atrophy.

BACKGROUND: Behr's syndrome is a classical phenotypic description of childhood-onset optic atrophy combined with various neurological symptoms, including ophthalmoparesis, nystagmus, spastic paraparesis, ataxia, peripheral neuropathy and learning difficulties. OBJECTIVE: Here we describe 4 patients with the classical Behr's syndrome phenotype from 3 unrelated families who carry homozygous nonsense mutations in the C12orf65 gene encoding a protein involved in mitochondrial translation. METHODS: Whole exome sequencing was performed in genomic DNA and oxygen consumption was measured in patient cell lines. RESULTS: We detected 2 different homozygous C12orf65 nonsense mutations in 4 patients with a homogeneous clinical presentation matching the historical description of Behr's syndrome. The first symptom in all patients was childhood-onset optic atrophy, followed by spastic paraparesis, distal weakness, motor neuropathy and ophthalmoparesis. CONCLUSIONS: We think that C12orf65 mutations are more frequent than previously suggested and screening of this gene should be considered not only in patients with mitochondrial respiratory chain deficiencies, but also in inherited peripheral neuropathies, spastic paraplegias and ataxias, especially with pre-existing optic atrophy.

Childhood-onset mitochondrial encephalomyopathies are severe, relentlessly progressive conditions. However, reversible infantile respiratory chain deficiency (RIRCD), due to a homoplasmic mt-tRNA(Glu) mutation, and reversible infantile hepatopathy, due to tRNA 5-methylaminomethyl-2-thiouridylate methyltransferase (TRMU) deficiency, stand out by showing spontaneous recovery, and provide the key to treatments of potential broader relevance. Modification of mt-tRNA(Glu) is a possible functional link between these two conditions, since TRMU is responsible for 2-thiouridylation of mt-tRNA(Glu), mt-tRNA(Lys) and mt-tRNA(Gln). Here we show that down-regulation of TRMU in RIRCD impairs 2-thiouridylation and exacerbates the effect of the mt-tRNA(Glu) mutation by triggering a mitochondrial translation defect in vitro. Skeletal muscle of RIRCD patients in the symptomatic phase showed significantly reduced 2-thiouridylation. Supplementation with l-cysteine, which is required for optimal TRMU function, rescued respiratory chain enzyme activities in human cell lines of patients with RIRCD as well as deficient TRMU. Our results show that l-cysteine supplementation is a potential treatment for RIRCD and for TRMU deficiency, and is likely to have broader application for the growing group of intra-mitochondrial translation disorders.

Highlights • We present an overview on mitochondrial protein synthesis. • We summarise nuclear factors involved in mitochondrial protein synthesis. • Tissue specific presentations highlight molecular mechanisms. • Altered modification of mt-tRNAs is a frequent cause of tissue specific presentations. • Interaction between cytosolic and mitochondrial translation needs further investigations., Defects of the mitochondrial protein synthesis cause a subgroup of mitochondrial diseases, which are usually associated with decreased activities of multiple respiratory chain (RC) enzymes. The clinical presentations of these disorders are often disabling, progressive or fatal, affecting the brain, liver, skeletal muscle, heart and other organs. Currently there are no effective cures for these disorders and treatment is at best symptomatic. The diagnosis in patients with multiple respiratory chain complex defects is particularly difficult because of the massive number of nuclear genes potentially involved in intra-mitochondrial protein synthesis. Many of these genes are not yet linked to human disease. Whole exome sequencing rapidly changed the diagnosis of these patients by identifying the primary defect in DNA, and preventing the need for invasive and complex biochemical testing. Better understanding of the mitochondrial protein synthesis apparatus will help us to explore disease mechanisms and will provide clues for developing novel therapies.

Exome sequencing identified compound heterozygous mutations in the recently discovered mitochondrial methionyl-tRNA formyltransferase (MTFMT) gene in two sisters with mild Leigh syndrome and combined respiratory chain deficiency. The mutations lead to undetectable levels of the MTFMT protein. Blue native polyacrylamide gel electrophoresis showed decreased complexes I and IV, and additional products stained with complex V antibodies, however the overall steady state level of mt-tRNAMet was normal. Our data illustrate that exome sequencing is an excellent diagnostic tool, and its value in clinical medicine is enormous, however it can only be optimally exploited if combined with detailed phenotyping and functional studies., Highlights • MTFMT mutations lead to mild Leigh syndrome and combined respiratory chain defect. • Blue native PAGE showed decreased complex I and IV but normal mt-tRNAMet. • Next generation sequencing (NGS) is a very powerful tool in mitochondrial disease. • It can be optimally exploited if combined with phenotyping and functional studies.

Periplakin regulates keratin organisation and participates in the assembly of epidermal cornified envelopes. A proteomic approach identified annexin A9 as a novel interacting partner for periplakin N-terminus. The presence of annexin A9 in complexes with periplakin was confirmed by immunoblotting of proteins immunoprecipitated by anti-HA or anti-annexin A9 antibodies. Both endogenous and GFP-tagged annexin A9 co-localise with endogenous periplakin and transfected periplakin N-terminus at MCF-7 cell borders and aggregate after Okadaic acid treatment. Annexin A9 and periplakin co-localise in the epidermis and annexin A9 is up-regulated in differentiating keratinocytes, but the epidermal annexin A9 expression does not require periplakin.Structured summary of protein interactionsAnnexin-9 physically interacts with Periplakin by anti bait co-immunoprecipitation (View interaction) Periplakin physically interacts with Annexin-9 by anti tag co-immunoprecipitation (View Interaction: 1, 2) Periplakin and Annexin-9 colocalize by fluorescence microscopy (View Interaction: 1, 2, 3) Keratin-8 and Periplakin colocalize by fluorescence microscopy (View interaction)

Collective migration of epithelial sheets requires maintenance of cell-cell junctions and co-ordination of the movement of the migrating front. We have investigated the role of keratin intermediate filaments and periplakin, a cytoskeletal linker protein, in the migration of simple epithelial cells. Scratch wounding induces bundling of keratins into a cable of tightly packed filaments adjacent to the free wound edge. Keratin re-organisation is preceded by a re-distribution of periplakin away from the free wound edge. Periplakin participates with dynamic changes in the keratin cytoskeleton via its C-terminal linker domain that co-localises with okadaic-acid-treated keratin granules. Stable expression of the periplakin C-terminal domain increases keratin bundling and Ser431 keratin phosphorylation at wound edge resulting in a delay in wound closure. Ablation of periplakin by siRNA inhibits keratin cable formation and impairs wound closure. Knockdown of keratin 8 with siRNA results in (1) a loss of desmoplakin localisation at cell borders, (2) a failure of MCF-7 epithelial sheets to migrate as a collective unit and (3) accelerated wound closure in vimentin-positive HeLa and Panc-1 cell lines. Thus, keratin 8 is required for the maintenance of epithelial integrity during migration and periplakin participates in the re-organisation of keratins in migrating cells.