Spectrin Repeats Research Articles

Composite biopolymer scaffolds shape muscle nucleus: Insights and perspectives from Drosophila.

Contractile muscle fibers produce enormous intrinsic forces during contraction/relaxation waves. These forces are directly applied to their cytoplasmic organelles including mitochondria, sarcoplasmic reticulum, and multiple nuclei. Data from our analysis of Drosophila larval somatic muscle fibers suggest that an intricate network of organized microtubules (MT) intermingled with Spectrin-Repeat-Containing Proteins (SRCPs) are major structural elements that protect muscle organelles and maintain their structure and position during muscle contraction. Whereas the perinuclear MT network provides structural rigidity to the myonucleus, the SRCPs Nesprin and Spectraplakin form semiflexible filamentous biopolymer networks, providing nuclei with the elasticity required to resist the contractile cytoplasmic forces produced by the muscle. Spectrin repeats are domains found in numerous structural proteins, which are able to unfold under tension and are subject to mechanical stresses in the cell. This unique composite scaffold combines rigidity and resilience in order to neutralize the oscillating cellular forces occurring during muscle contraction/relaxation waves and thereby protect myonuclei. We suggest that the elastic properties of SRCPs are critical for nuclear protection and proper function in muscle fibers.

The Physiological Molecular Shape of Spectrin: A Compact Supercoil Resembling a Chinese Finger Trap.

The primary, secondary, and tertiary structures of spectrin are reasonably well defined, but the structural basis for the known dramatic molecular shape change, whereby the molecular length can increase three-fold, is not understood. In this study, we combine previously reported biochemical and high-resolution crystallographic data with structural mass spectroscopy and electron microscopic data to derive a detailed, experimentally-supported quaternary structure of the spectrin heterotetramer. In addition to explaining spectrin’s physiological resting length of ~55-65 nm, our model provides a mechanism by which spectrin is able to undergo a seamless three-fold extension while remaining a linear filament, an experimentally observed property. According to the proposed model, spectrin’s quaternary structure and mechanism of extension is similar to a Chinese Finger Trap: at shorter molecular lengths spectrin is a hollow cylinder that extends by increasing the pitch of each spectrin repeat, which decreases the internal diameter. We validated our model with electron microscopy, which demonstrated that, as predicted, spectrin is hollow at its biological resting length of ~55-65 nm. The model is further supported by zero-length chemical crosslink data indicative of an approximately 90 degree bend between adjacent spectrin repeats. The domain-domain interactions in our model are entirely consistent with those present in the prototypical linear antiparallel heterotetramer as well as recently reported inter-strand chemical crosslinks. The model is consistent with all known physical properties of spectrin, and upon full extension our Chinese Finger Trap Model reduces to the ~180-200 nm molecular model currently in common use.

A Mutation in Syne2 Causes Early Retinal Defects in Photoreceptors, Secondary Neurons, and Müller Glia.

The purpose of this study was to identify the molecular basis and characterize the pathological consequences of a spontaneous mutation named cone photoreceptor function loss 8 (cpfl8) in a mouse model with a significantly reduced cone electroretinography (ERG) response. The chromosomal position for the recessive cpfl8 mutation was determined by DNA pooling and by subsequent genotyping with simple sequence length polymorphic markers in an F2 intercross phenotyped by ERG. Genes within the candidate region of both mutants and controls were directly sequenced and compared. The effects of the mutation were examined in longitudinal studies by light microscopy, marker analysis, transmission electron microscopy, and ERG. The cpfl8 mutation was mapped to Chromosome 12, and a premature stop codon was identified in the spectrin repeat containing nuclear envelope 2 (Syne2) gene. The reduced cone ERG response was due to a significant reduction in cone photoreceptors. Longitudinal studies of the early postnatal retina indicated that the cone photoreceptors fail to develop properly, rod photoreceptors mislocalize to the inner nuclear layer, and both rods and cones undergo apoptosis prematurely. Moreover, we observed migration defects of secondary neurons and ectopic Müller cell bodies in the outer nuclear layer in early postnatal development. SYNE2 is important for normal retinal development. We have determined that not only is photoreceptor nuclear migration affected, but also the positions of Müller glia and secondary neurons are disturbed early in retinal development. The cpfl8 mouse model will serve as an important resource for further examining the role of nuclear scaffolding and migration in the developing retina.

Domain repeats related to innate immunity in the South African abalone, Haliotis midae

Molluscs predominately use the cellular defence system as the primary mechanism of defence against pathogenic infection, in which haemocytes play a pivotal role. Haliotis midae is a commercially important South African species that it is susceptible to bacterial pathogens, fungal and yeast infections in the farming environment. The current study aims to enrich the current knowledge regarding H. midae innate immunity by investigating the presence and evolution of domain repeats. The bioinformatics approach used in this study, detected five repeat families in the H. midae transcriptome. These repeats families include mixed alpha and beta (leucine-rich and ankyrin), spectrin repeats, beta-propellers (WD40) and alfa-structure repeat (TPR-like). The expansion of key gene families related to host defence may be important to abalone adaptation to life in a pathogen-rich environment.

Supervillin Binds the Rac/Rho‐GEF Trio and Increases Trio‐mediated Rac1 Activation

We investigated cross‐talk between the membrane‐associated, myosin II‐regulatory protein supervillin and the actin‐regulatory small GTPases Rac1, RhoA, and Cdc42. Supervillin knockdown reduced Rac1‐GTP loading, but not the GTP loading of RhoA or Cdc42, in HeLas cells with normal levels of the Rac1‐activating protein Trio. No reduction in Rac1‐GTP loading was observed when supervillin levels were reduced in Trio‐depleted cells. Conversely, overexpression of supervillin isoform 1 (SV1) or, especially, isoform 4 (SV4) increased Rac1 activation. Inhibition of the Trio‐mediated Rac1 guanine nucleotide exchange (GEF) activity with ITX3 partially blocked the SV4‐mediated increase in Rac1‐GTP. Both SV4 and SV1 co‐localized with Trio at or near the plasma membrane in ruffles and cell surface projections. Two sequences within supervillin bound directly to Trio spectrin repeats 4‐7: SV1‐171, which contains N‐terminal residues found in both SV1 and SV4 and the SV4‐specific differentially spliced coding exons 3, 4, and 5 within SV4 (SV4‐E345; SV4 amino acids 276 – 669). In addition, SV4‐E345 interacted with the homologous sequence in rat kalirin (repeats 4‐7, amino acids 531 ‐ 1101). Overexpressed SV1‐174 and SV4‐E345 affected Rac1‐GTP loading, but only in cells with endogenous levels of Trio. Trio residues 771 – 1057, which contain both supervillin‐interaction sites, exerted a dominant‐negative effect on cell spreading. Supervillin and Trio knockdowns, separately or together, inhibited cell spreading, suggesting that supervillin regulates the Rac1 guanine nucleotide exchange activity of Trio, and potentially also kalirin, during cell spreading and lamellipodia extension.

Spectrin repeat containing nuclear envelope 1 and forkhead box protein E1 are promising markers for the detection of colorectal cancer in blood.

Identifying biomarkers in body fluids may improve the noninvasive detection of colorectal cancer. Previously, we identified N-Myc downstream-regulated gene 4 (NDRG4) and GATA binding protein 5 (GATA5) methylation as promising biomarkers for colorectal cancer in stool DNA. Here, we examined the utility of NDRG4, GATA5, and two additional markers [Forkhead box protein E1 (FOXE1) and spectrin repeat containing nuclear envelope 1 (SYNE1)] promoter methylation as biomarkers in plasma DNA. Quantitative methylation-specific PCR was performed on plasma DNA from 220 patients with colorectal cancer and 684 noncancer controls, divided in a training set and a test set. Receiver operating characteristic analysis was performed to measure the area under the curve of GATA5, NDRG4, SYNE1, and FOXE1 methylation. Functional assays were performed in SYNE1 and FOXE1 stably transfected cell lines. The sensitivity of NDRG4, GATA5, FOXE1, and SYNE1 methylation in all stages of colorectal cancer (154 cases, 444 controls) was 27% [95% confidence interval (CI), 20%-34%), 18% (95% CI, 12%-24%), 46% (95% CI, 38%-54%), and 47% (95% CI, 39%-55%), with a specificity of 95% (95% CI, 93%-97%), 99% (95% CI, 98%-100%), 93% (95% CI, 91%-95%), and 96% (95% CI, 94%-98%), respectively. Combining SYNE1 and FOXE1, increased the sensitivity to 56% (95% CI, 48%-64%), while the specificity decreased to 90% (95% CI, 87%-93%) in the training set and to 58% sensitivity (95% CI, 46%-70%) and 91% specificity (95% CI, 80%-100%) in a test set (66 cases, 240 controls). SYNE1 overexpression showed no major differences in cell proliferation, migration, and invasion compared with controls. Overexpression of FOXE1 significantly decreased the number of colonies in SW480 and HCT116 cell lines. Overall, our data suggest that SYNE1 and FOXE1 are promising markers for colorectal cancer detection.

SPTAN1 encephalopathy: distinct phenotypes and genotypes.

Recent progress in genetic analysis reveals that a significant proportion of cryptogenic epileptic encephalopathies are single-gene disorders. Mutations in numerous genes for early-onset epileptic encephalopathies have been rapidly identified, including in SPTAN1, which encodes α-II spectrin. The aim of this review is to delineate SPTAN1 encephalopathy as a distinct clinical syndrome. To date, a total of seven epileptic patients with four different in-frame SPTAN1 mutations have been identified. The major clinical features of SPTAN1 mutations include epileptic encephalopathy with hypsarrhythmia, no visual attention, acquired microcephaly, spastic quadriplegia and severe intellectual disability. Brainstem and cerebellar atrophy and cerebral hypomyelination, as observed by magnetic resonance imaging, are specific hallmarks of this condition. A milder variant is characterized by generalized epilepsy with pontocerebellar atrophy. Only in-frame SPTAN1 mutations in the last two spectrin repeats in the C-terminal region lead to dominant negative effects and these specific phenotypes. The last two spectrin repeats are required for α/β spectrin heterodimer associations and the mutations can alter heterodimer formation between the two spectrins. From these data we suggest that SPTAN1 encephalopathy is a distinct clinical syndrome owing to specific SPTAN1 mutations. It is important that this syndrome is recognized by pediatric neurologists to enable proper diagnostic work-up for patients.

Proteins in Striated Muscles that Transcribed from the Contiguous Region of Connectin Gene

Connectin is the largest protein that connects between Z-line and M-line of sarcomere and functions as a molecular spring of vertebrate striated muscles. At the contiguous region of connectin gene on mammalian genomes, there are two genes for proteins that function remain unknown. One protein (about 75kDa) consists of a SEC14 domain and three spectrin repeats, and the other protein (about 150kDa) consists of six spectrin repeats and an immunogloburin-like domain. We performed the RT-PCR experiments and revealed that these two genes are expressed in striated muscles. The western blot tests using newly produced antibodies also indicated that these proteins existed in striated muscles. The immunofluorescence microscopic observation elucidated that the 75kDa protein are located on the membrane of muscle fiber but the 150 kDa protein are localized at the Z-line of the sarcomere. The localization of these proteins are also confirmed by the transfection of GFP-fusion proteins into cultured skeletal muscle cells. These insights indicate that novel proteins that transcribed from the contiguous genes of connectin gene exist in striated muscles.

Supervillin binds the Rac/Rho-GEF Trio and increases Trio-mediated Rac1 activation.

We investigated cross-talk between the membrane-associated, myosin II-regulatory protein supervillin and the actin-regulatory small GTPases Rac1, RhoA, and Cdc42. Supervillin knockdown reduced Rac1-GTP loading, but not the GTP loading of RhoA or Cdc42, in HeLa cells with normal levels of the Rac1-activating protein Trio. No reduction in Rac1-GTP loading was observed when supervillin levels were reduced in Trio-depleted cells. Conversely, overexpression of supervillin isoform 1 (SV1) or, especially, isoform 4 (SV4) increased Rac1 activation. Inhibition of the Trio-mediated Rac1 guanine nucleotide exchange activity with ITX3 partially blocked the SV4-mediated increase in Rac1-GTP. Both SV4 and SV1 co-localized with Trio at or near the plasma membrane in ruffles and cell surface projections. Two sequences within supervillin bound directly to Trio spectrin repeats 4-7: SV1-171, which contains N-terminal residues found in both SV1 and SV4 and the SV4-specific differentially spliced coding exons 3, 4, and 5 within SV4 (SV4-E345; SV4 amino acids 276-669). In addition, SV4-E345 interacted with the homologous sequence in rat kalirin (repeats 4-7, amino acids 531-1101). Overexpressed SV1-174 and SV4-E345 affected Rac1-GTP loading, but only in cells with endogenous levels of Trio. Trio residues 771-1057, which contain both supervillin-interaction sites, exerted a dominant-negative effect on cell spreading. Supervillin and Trio knockdowns, separately or together, inhibited cell spreading, suggesting that supervillin regulates the Rac1 guanine nucleotide exchange activity of Trio, and potentially also kalirin, during cell spreading and lamellipodia extension.

RACK1 (receptor for activated C-kinase 1) interactions with spectrin repeat elements.

Receptor for activated C-kinase 1 (RACK1) is an intracellular scaffolding protein involved in a multitude of signalling pathways. The cytoskeleton is fundamental for intracellular cell signalling as it forms an interconnected network of regulatory proteins. Here, spectrin is a central component as it forms the actin-spectrin network that serves as docking surfaces for cellular components. The interaction between RACK1 and components of spectrin, the single spectrin repeats R16, R17 and the double spectrin repeat R1617 from the α-spectrin chain were investigated by biosensor technology and docking analysis. RACK1 associated only weakly to R16 (KD = 1.0 ± 0.5 × 10(-6) M), about 20 times stronger to R1617 (KD = 5.3 ± 0.7 × 10(-8) M) and 100 times stronger to R17 (KD = 0.9 ± 0.3 × 10(-8) M). Docking analysis showed that while R16 alone preferentially docked with its B-helix, R17 docked through its A-helix and BC loop. The double repeat and RACK1 mainly formed two different complex conformations. R1617 docked tangentially to the N/C-terminal of RACK1 or radially along a groove on the outer surface of RACK1. These configurations could account for the slight increase in entropic and the decrease in enthalpic interactions for the R1617-RACK1 interaction, compared with the interactions of RACK1 to the two single repeats. Our results suggest a mode of interaction that allows spectrin to attach to the N/C part of RACK through the inter-helical AB and BC loops and adopt a multitude of configurations in between the two limiting configurations.

Severe Alpha-Spectrin Linked Recessive Hereditary Spherocytosis

Approximately one quarter of cases of hereditary spherocytosis exhibit autosomal recessive inheritance. Patients with recessive hereditary spherocytosis (rHS) are more severely affected than patients with typical, dominant HS. Many rHS patients present in infancy with life-threatening hemolytic anemia; some are transfusion-dependent (TD). Erythrocytes from most rHS patients are spectrin deficient, leading to destabilization of the lipid bilayer. In most cases, the genetic defects in the SPTA1 gene leading to rHS are unknown. We studied 19 families with rHS suspected by clinical and laboratory data including 5 of the original rHS kindreds described by Agre et al. Probands from 9 of the 19 families were severely affected, demonstrating life-long transfusion dependence. After obtaining a genetic diagnosis, two probands were splenectomized and became transfusion independent. In the remaining 10 families, most patients had a history of receiving blood transfusions, particularly during infancy. Most underwent early splenectomy that ameliorated but did not cure their anemia. Hematocrits of affected patients postsplenectomy ranged from 21-51%. In untransfused patients, biochemical analyses included SDS-PAGE of erythrocyte membrane proteins. Quantitative analyses of spectrin content, measured by spectrin/band 3 ratios, demonstrated spectrin deficiency from 43-78% in rHS erythrocyte membranes. Genetic studies included Sanger sequencing (6 families) and/or whole exome analyses (WES, 13 families). Genomic DNA was amplified using primers flanking the 52 coding exons and promoter region of the SPTA1 gene, followed by Sanger sequencing analysis of the amplicons. Alternatively, genomic DNA was subjected to WES via targeted exon capture followed by ultrahigh throughput sequencing of captured DNA. Sequencing data were mapped and variants called by the GATK algorithm. Variants were filtered then further assessed via conservation and mutation prediction programs. Numerous unique null SPTA1 alleles were found. Two TD patients had deleterious mutations in both SPTA1 alleles; one with nonsense mutations in trans died of liver failure associated with transfusion-related iron overload, the other with nonsense and splicing mutations in trans remains transfusion dependent. Additional null alleles included 6 nonsense, 4 splicing, and 3 frameshift mutations. These null alleles were frequently in trans to novel missense mutations. Eight unique missense mutations (in spectrin repeats 2, 4 and 14) were identified in conserved residues and all were predicted to be deleterious by mutation prediction algorithms. Two missense mutations were found in multiple families (repeat 14 in 4 families, repeat 2 in 2 families). Two TD patients were homozygous for missense mutations located in critical residues of the alpha-beta spectrin self-association domain. One of these patients had a sibling homozygous for the same mutation die in the perinatal period due to complications of anemia. The repeat 2 mutation was found in the heterozygous state in 3 other rHS kindreds in trans to 3 different null alleles. Probands from two kindreds suffered from homozygous missense mutations in conserved residues that were predicted to be deleterious by mutation prediction algorithms (repeat 2 and repeat 14). In 7 alpha-spectrin linked rHS probands, only a single deleterious allele was identified. Erythrocyte membranes from 6 of these patients (the seventh was TD) exhibited severe spectrin deficiency, indicating that another mutation associated with a production-defective SPTA1 allele was present in trans. In summary, 27 novel mutations were identified on 38 SPTA1 alleles. The data demonstrate how the synthesis of complementary lines of investigation, i.e. clinical, laboratory, biochemical and genetic data, can be leveraged to define and advance our understanding of inherited hematologic disease. They also demonstrate the significant genetic heterogeneity in hereditary spherocytosis and support the use of genomic strategies for diagnosis, particularly in severe cases, allowing design of appropriate therapy. DisclosuresNo relevant conflicts of interest to declare.

G.P.147: Two novel epsilon isoforms of nesprin-2, a protein linked to Emery–Dreifuss muscular dystrophy

Nesprin proteins have a central rod domain of spectrin repeats and are intracellular linkers and scaffolds. Full length or "giant" nesprin-1 and nesprin-2 have N-terminal calponin homology domains that bind the actin cytoskeleton and C-terminal transmembrane KASH domains which anchor the nesprins to the outer nuclear membrane. A number of short isoforms of nesprin-1 and nesprin-2 are produced by internal promotion and by alternative splicing. Mutations in nesprin-1 and nesprin-2 can cause Emery-Dreifuss muscular dystrophy and dilated cardiomyopathy. Nesprin-1-alpha-2 is found almost exclusively in skeletal muscle and heart and all the known mutations in nesprins that are associated with EDMD or DCM lie within the alpha isoform sequence. Two novel "epsilon" isoforms of nesprin-2 were predicted by bioinformatics and we have demonstrated the existence of corresponding proteins for the first time. Because they are similar to nesprin-1 alpha in size and structure, we evaluated the significance of the epsilon isoforms in 20 human tissues and 7 human cell lines using qPCR. N2-epsilon-1 was expressed only in ovary and early embryonic cells (Ntera-2 and ESC), while N2-epsilon-2 was expressed in several mature tissues, including cardiac, but not skeletal, muscle. Western blotting confirmed the presence of epsilon-1 protein in Ntera-2 and ESC and epsilon-2 protein in heart and brain. Total nesprin transcript in ESC was mainly nesprin-2-giant (77%) and nesprin-2-epsilon-1 (21%). PCR indicated that most of the nesprin-2 mRNA in ESC lacked the KASH domain, whereas heart and skeletal muscle appeared to contain high levels of nesprin-2 KASH. Immunofluorescence microscopy with a monoclonal antibody against nesprin-2 showed nuclear rim staining in heart and skeletal muscle sections, but in KASH-less ESC there was an intense nucleoplasmic speckle-like distribution. Supported by British Heart Foundation (PG/11/71/29091).

Mammalian microtubule P-body dynamics are mediated by nesprin-1.

Nesprins are a multi-isomeric family of spectrin-repeat (SR) proteins, predominantly known as nuclear envelope scaffolds. However, isoforms that function beyond the nuclear envelope remain poorly examined. Here, we characterize p50(Nesp1), a 50-kD isoform that localizes to processing bodies (PBs), where it acts as a microtubule-associated protein capable of linking mRNP complexes to microtubules. Overexpression of dominant-negative p50(Nesp1) caused Rck/p54, but not GW182, displacement from microtubules, resulting in reduced PB movement and cross talk with stress granules (SGs). These cells disassembled canonical SGs induced by sodium arsenite, but not those induced by hydrogen peroxide, leading to cell death and revealing PB-microtubule attachment is required for hydrogen peroxide-induced SG anti-apoptotic functions. Furthermore, p50(Nesp1) was required for miRNA-mediated silencing and interacted with core miRISC silencers Ago2 and Rck/p54 in an RNA-dependent manner and with GW182 in a microtubule-dependent manner. These data identify p50(Nesp1) as a multi-functional PB component and microtubule scaffold necessary for RNA granule dynamics and provides evidence for PB and SG micro-heterogeneity.

Spectraplakins

Spectraplakins

Nonenzymatic domains of Kalirin7 contribute to spine morphogenesis through interactions with phosphoinositides and Abl

Like several Rho GDP/GTP exchange factors (GEFs), Kalirin7 (Kal7) contains an N-terminal Sec14 domain and multiple spectrin repeats. A natural splice variant of Kalrn lacking the Sec14 domain and four spectrin repeats is unable to increase spine formation; our goal was to understand the function of the Sec14 and spectrin repeat domains. Kal7 lacking its Sec14 domain still increased spine formation, but the spines were short. Strikingly, Kal7 truncation mutants containing only the Sec14 domain and several spectrin repeats increased spine formation. The Sec14 domain bound phosphoinositides, a minor but crucial component of cellular membranes, and binding was increased by a phosphomimetic mutation. Expression of KalSec14-GFP in nonneuronal cells impaired receptor-mediated endocytosis, linking Kal7 to membrane trafficking. Consistent with genetic studies placing Abl, a non-receptor tyrosine kinase, and the Drosophila orthologue of Kalrn into the same signaling pathway, Abl1 phosphorylated two sites in the fourth spectrin repeat of Kalirin, increasing its sensitivity to calpain-mediated degradation. Treating cortical neurons of the wild-type mouse, but not the Kal7(KO) mouse, with an Abl inhibitor caused an increase in linear spine density. Phosphorylation of multiple sites in the N-terminal Sec14/spectrin region of Kal7 may allow coordination of the many signaling pathways contributing to spine morphogenesis.

Nesprin-1 role in DNA damage response

Nuclear envelope (NE) proteins have fundamental roles in maintaining nuclear structure, cell signaling, chromatin organization, and gene regulation, and mutations in genes encoding NE components were identified as primary cause of a number of age associated diseases and cancer. Nesprin-1 belongs to a family of multi-isomeric NE proteins that are characterized by spectrin repeats. We analyzed NE components in various tumor cell lines and found that Nesprin-1 levels were strongly reduced associated with alterations in further NE components. By reducing the amounts of Nesprin-1 by RNAi mediated knockdown, we could reproduce those alterations in mouse and human cell lines. In a search for novel Nesprin-1 binding proteins, we identified MSH2 and MSH6, proteins of the DNA damage response pathway, as interactors and found alterations in the corresponding pathways in cells with lower Nesprin-1 levels. We also noticed increased number of γH2AX foci in the absence of exogenous DNA damage as was seen in tumor cells. The levels of phosphorylated kinases Chk1 and 2 were altered in a manner resembling tumor cells and the levels of Ku70 were low and the protein was not recruited to the DNA after hydroxyurea (HU) treatment. Our findings indicate a role for Nesprin-1 in the DNA damage response pathway and propose Nesprin-1 as novel player in tumorigenesis and genome instability.

Localization and Binding Partners of SESTD1 in Skeletal Muscles

Myofibrillogenesis is a complex production system of the striated muscles, which are correctly built by a lot of structural and signaling proteins. Some insights about the molecular mechanism of myofibrillogenesis were elucidated but most of them are not uncovered. SESTD1 is a novel protein that consists of SEC14 domain, three spectrin repeats and unique sequences and is expressed in various tissues including striated muscles. One research suggests that SESTD1 is involved in the planar cell polarity pathway during mammalian embryonic development and another research suggests that SESTD1 regulates some transient receptor potential channels in smooth muscle. However, it remains unclear about the function of SESTD1 in striated muscles. In this research, we investigated localization and binding partner of SESTD1 in striated muscles. SESTD1 was detected in the whole extracts from skeletal but not cardiac muscles by western blot using anti-SESTD1 monoclonal antibody. Immunofluorescence microscopy using the antibody revealed that SESDT1 is localized at Z-line of sarcomere. To search the binding partners of SESTD1, we carried out yeast two-hybrid screening with skeletal muscle cDNA libraries. Several proteins located in Z-line were obtained as candidates for SESTD1 C-terminus binding proteins and alpha-actin was obtained as a SESTD1 N-terminus binding protein. These results indicate that SESTD1 is the novel Z-line protein of the sarcomere in skeletal muscle and may function through binding to both Z-line structural proteins and actin filaments. We are currently investigating the role of SESTD1 in Z-line formation and actin filament assembly.

The Cellular Content of Non-Erythroid Spectrins and Ankyrins is Modulated by External Forces

Cells are exposed to mechanical forces in the form of stretch, shear stress, and hydro-static pressure and they transduce those forces into physical as well as biochemical responses. An important, mechanical cell-component that also takes part in this process of mechano-transduction is the spectrin based membrane skeleton. In the context of non-erythroid cells this structure has seen very little attention so far, while its counterpart in erythroid cells, which lack any other significant mechanical structure, has been extensively studied. For example, it was investigated that the erythroid α-spectrin has an E2/E3 ubiquitination site that allows it to either ubiquitinate itself or spectrin associated Ankyrin. Interestingly, the corresponding domain in non-erythroid α-Spectrins is both conserved and is located at a mechano-sensitive site within a tri-helical spectrin repeat. Thus, we looked at the change in non-erythroid Spectrins' and Ankyrins', suspected targets' contents in NIH-3T3 Fibroblasts and multipotent-10T1/2 cells as they are exposed to stretch magnitudes of 20% at a frequency of 1Hz. Using a combination of fluorescence microscopy, quantitative gel analysis and immunoprecipitation, we find that spectrin content drastically decreases by up to 80%, while ankyrin content increases by up to 30% in the two cell types when they are exposed to bi-axial stretch. Technically, we were also able to establish that quantitative fluorescence microscopy on immunostained fixed cells is as suitable to measure relative changes in protein content as immunoprecipitated Coomassie blue stained protein gels. Finally, our results from co-immunoprecipitation and Western blotting indeed suggest that the cause of these changes in protein content is the poly-ubiqutination activity of spectrin on itself as well as ankyrin. Since both proteins act also as scaffolds for membrane associated proteins, these force induced changes should also impact membrane organization and structure.

Spectrin: Structure, function and disease

Spectrin is a large, cytoskeletal, and heterodimeric protein composed of modular structure of α and β subunits, it typically contains 106 contiguous amino acid sequence motifs called "spectrin repeats". Spectrin is crucial for maintaining the stability and structure of the cell membrane and the shape of a cell. Moreover, it contributes to diverse cell functions such as cell adhesion, cell spreading, and the cell cycle. Mutations of spectrin lead to various human diseases such as hereditary hemolytic anemia, type 5 spinocerebellar ataxia, cancer, as well as others. This review focuses on recent advances in determining the structure and function of spectrin as well as its role in disease.

Mutations in LMNA Modulate the Lamin A - Nesprin-2 Interaction and Cause LINC Complex Alterations

BackgroundIn eukaryotes the genetic material is enclosed by a continuous membrane system, the nuclear envelope (NE). Along the NE specific proteins assemble to form meshworks and mutations in these proteins have been described in a group of human diseases called laminopathies. Laminopathies include lipodystrophies, muscle and cardiac diseases as well as metabolic or progeroid syndromes. Most laminopathies are caused by mutations in the LMNAgene encoding lamins A/C. Together with Nesprins (Nuclear Envelope Spectrin Repeat Proteins) they are core components of the LINC complex (Linker of Nucleoskeleton and Cytoskeleton). The LINC complex connects the nucleoskeleton and the cytoskeleton and plays a role in the transfer of mechanically induced signals along the NE into the nucleus, and its components have been attributed functions in maintaining nuclear and cellular organization as well as signal transduction.ResultsHere we narrowed down the interaction sites between lamin A and Nesprin-2 to aa 403–425 in lamin A and aa 6146–6347 in Nesprin-2. Laminopathic mutations in and around the involved region of lamin A (R401C, G411D, G413C, V415I, R419C, L421P, R427G, Q432X) modulate the interaction with Nesprin-2 and this may contribute to the disease phenotype. The most notable mutation is the lamin A mutation Q432X that alters LINC complex protein assemblies and causes chromosomal and transcription factor rearrangements.ConclusionMutations in Nesprin-2 and lamin A are characterised by complex genotype phenotype relations. Our data show that each mutation in LMNAanalysed here has a distinct impact on the interaction among both proteins that substantially explains how distinct mutations in widely expressed genes lead to the formation of phenotypically different diseases.