Spectrin-like Protein Research Articles

Historical aspects of muscle research in the Dubowitz Neuromuscular Centre: the Hammersmith days

Historical aspects of muscle research in the Dubowitz Neuromuscular Centre: the Hammersmith days

Grl1 Protein is a Candidate K Antigen in Tetrahymena thermophila

In Tetrahymena, K antigens associate only with mature basal bodies and are expected to play important roles in the morphogenesis and function of the membrane skeleton around basal bodies, but these proteins have not been identified and their functions are unknown. Commercially available anti-human Rho GDP-dissociation inhibitor α (RhoGDIα) antibody (sc-33201) was accidentally found to show very similar immunofluorescence staining patterns to those of anti-K antigen antibodies, such as 424A8 and 10D12 mouse monoclonal antibodies, in Tetrahymena. A 40kDa protein recognized by this antibody was partially purified and identified as granule lattice protein 1 (Grl1p) by matrix-assisted laser desorption/ionization-tandem time-of-flight mass spectrometry. In immunoblotting experiments this antibody was suggested to recognize endogenous Grl1p. The three-dimensional structure of proGrl1p protein predicted by I-TASSER was similar to a spectrin family protein. Grl1 may be a K antigen and a spectrin-like protein in Tetrahymena.

ADVANCES IN MOLECULAR THERAPY RESEARCH ON DYSTROPHIN-DEFICIENT MUSCULAR DYSTROPHY

Duchenne muscular dystrophy (DMD) is a lethal muscle disorder caused by mutations of the DMD gene, which encodes a 427-kDa spectrin-like cytoskeletal protein, dystrophin. Exon skipping induced by antisense oligonucleotides is a novel method to restore the reading frame of the mutated DMD gene and rescue dystrophin expression. We recently demonstrated that systemic delivery of antisense phosphorodiamidate morpholino oligonucleotides (PMOs) targeting exons 6 and 8 of the canine DMD gene efficiently recovered functional dystrophin at the sarcolemma of dystrophic dogs, and improved performance of the affected dogs without serious side effects. As a strategy to target hot spots of mutation in the DMD gene, we also tried exon 51-skipping using PMOs in mdx52 mice to convert an out-of-frame mutation into an in-frame mutation with restoration of dystrophin expression in various muscles and improvement of pathology and function. Progress in adeno-associated virus vector serotype 9 (AAV-9)-mediated DMD gene therapy has enabled the delivery of the therapeutic gene to the whole musculature, including cardiac muscle, while evoking minimal immunological reactions in mice, dogs, and non-human primates. Furthermore, DMD-derived patient-specific induced pluripotent stem (iPS) cells could be a potential source for cell therapy, although there are at present hurdles to be overcome. In the future, this technology could be used in combination with exon skipping or AAV-mediated gene therapy to achieve clinical benefits.

The 8th and 9th tandem spectrin-like repeats of utrophin cooperatively form a functional unit to interact with polarity-regulating kinase PAR-1b

Utrophin is a widely expressed paralogue of dystrophin, the protein responsible for Duchenne muscular dystrophy. Utrophin is a large spectrin-like protein whose C-terminal domain mediates anchorage to a laminin receptor, dystroglycan (DG). The rod domain, composed of 22 spectrin-like repeats, connects the N-terminal actin-binding domain and the C-terminal DG binding domain, and thus mediates molecular linkage between intracellular F-actin and extracellular basement membrane. Previously, we demonstrated that a cell polarity-regulating kinase, PAR-1b, interacts with the utrophin–DG complex, and positively regulates the interaction between utrophin and DG. In this study, we demonstrate that the 8th and 9th spectrin-like repeats (R8 and R9) of utrophin cooperatively form a PAR-1b-interacting domain, and that Ser1258 within R9 is specifically phosphorylated by PAR-1b. Substitution of Ser1258 to alanine reduces the interaction between utrophin and DG, suggesting that the Ser1258 phosphorylation contributes to the stabilization of the utrophin–DG complex. Interestingly, PAR-1b also binds and phosphorylates R8–9 of dystrophin, and colocalizes with dystrophin at the skeletal muscle membrane. These results reveal a novel function of the rod domain of utrophin beyond that of a passive structural linker connecting the N- and C-terminal domain.

Proteolytic Cleavage of a Spectrin-Related Protein by Calcium-Dependent Protease in Neurospora crassa

To investigate the functional significance of a cytoskeletal spectrin-like protein, we studied its localization pattern in Neurospora crassa and sought the answer to whether it is a substrate for another apically localized protein, the calcium-dependent protease (CDP II). Immunoblots of crude extracts from exponentially growing mycelia, separated by one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis using antichicken alpha/beta-spectrin antibodies, revealed a single band of approximately relative mass (Mr) 100 kDa with an isoeletric point (pI) in the range of 6.5 to 7.0. Despite rigorous efforts, we could not confirm the presence of an Mr 240- to 220-kDa spectrin-like protein in N. crassa. The immunofluorescence- and immunogold-labeling Mr 100-kDa protein showed its predominance along the plasma membrane of the conidia during the swelling phase of germination. In contrast, in the germ tubes and the growing hyphae, the localization was polarized and concentrated mainly in the apical region. The in vitro proteolysis experiments showed that indeed this protein is a preferred substrate of CDP II which is, as mentioned previously, also localized in the apical regions of the hyphae. These results indicate a putative functional relationship between these two proteins (spectrin-like protein and CDP II) in the dynamics of tip growth.

Rearrangement of the actin-spectrin cytoskeleton during oocyte maturation of the starfish Asterias amurensis

Actin and spectrin localization in the oocytes of the starfish Asterias amurensis at hormonal induction of maturation until the destruction of the germinal vesicular membrane has been investigated by immunocytochemical and immunoblotting methods. In immature oocytes, spectrinlike protein and actin are detected to be colocalized in the undermembranous area of the cytoplasm and nuclear membrane. 1-Methyladenine causes redistribution of these proteins into intracellular structures. The actin-spectrin cytoskeleton rearrangement is shown to start at the animal pole of the oocyte and to spread then to its vegetative pole.

Active Kinase Proteome Screening Reveals Novel Signal Complexity in Cardiomyopathy

Recent advances in the characterization of the phosphoproteome have been limited to measuring phosphorylation statuses, which imply but do not measure protein kinase activity directly. As such, the ability to screen, compare, and define multiple protein enzymatic activities across divergent samples remains a daunting challenge in proteomics. Here, we describe a gel-based kinase assay coupled to MS identification as an approach to map global kinase activity and assign pathway architecture to specified biologic contexts. We demonstrate the utility of this method as a platform for the comparison of proteomes based on differences in both kinase activities and for use in the de novo substrate identification for individual kinases. This approach allowed us to map the signal perturbations in the post-natal heart that were associated with activation of a myopathic cascade as mediated by the mitogen-activated protein kinase MKK6 and established the novel observation that MKK6 promotes the development of cardiomyopathy through multiple substrate interactions.

Gravisensing in single-celled systems – update on characean rhizoids and protonemata

Single-celled and tip-growing rhizoids and protonemata of the characean algae have been intensively studied and there is considerable progress in the understanding of the molecular and cellular mechanisms underlying gravisensing and gravity-dependent growth. In higher plant statocytes, the role of actin in both processes is still a matter of intense debate, but there is clear evidence that actin coordinates both processes in characean rhizoids and protonemata. The multiple functions and dynamic nature of the actin cytoskeleton in these cells are based on the concerted action of a variety of actin-binding proteins. Profilin, actin-depolymerizing factor, a spectrin-like protein, villin and fimbrin have been detected which control apical actin polymerization and regulate the dynamic remodeling of the actin arrangement. An actomyosin-based system was shown to (i) mediate the transport of secretory vesicles to the growing tip, (ii) establish the incorporation of cell wall material and (iii) coordinate the tip-focussed distribution of calcium channels which establish the tip-high calcium gradient for local exocytosis. Experiments performed in microgravity have shown that the actomyosin system precisely coordinates the position of statoliths in rhizoids and protonemata and, upon a change in orientation, directs sedimenting statoliths to specific areas at the plasma membrane where physical contact with gravisensor molecules initiates growth reorientation. The upward growth response of protonemata was shown to be preceded by a statolith-induced and actin-dependent relocalization of the Ca 2+-gradient to the upper flank that does not occur in positively gravitropic rhizoids, in which sedimented statoliths cause differential growth of the opposite subapical cell flank. Thus, constant actin polymerization in the growing tip and the spatiotemporal control of actin remodeling by numerous actin-binding proteins are essential for gravity sensing and polarized growth of characean rhizoids and protonemata.

Identification and function analysis of spectrin-like protein in pollen tubes of lily (Lilium davidii Duch)

The elongation of pollen tube is an important process of sexual reproduction in higher plant. Cytoskeleton plays a major regulatory role in the elongation of pollen tubes. But whether membrane skeleton is involved in the pollen tube elongation is not clear. In this study, immunochemical detection of spectrin-like protein has been carried out in pollen tubes. By use of 2-dimensional electrophoresis(2DE) and western blotting, two spectrin-like proteins are found, one is 150 kD, and the other is 105 kD, with pI being 4.54 and 4.39, respectively. 150 kD spectrin-like protein is located in plasma membrane of pollen tube and 105 kD spectrin-like protein is located in cytoplasm, probably functioning as a subunit to form a dimmer (210 kD)in vivo. The elongation of pollen tubes is inhibited after spectrin antibody was injected into a growing pollen tube. These results suggest that spectrin-like proteins exist in pollen tube and play an important regulating role in the elongation process of pollen tubes from lily.

Signal transduction through the Gal?GalNAc lectin of Entamoeba histolytica involves a spectrin-like protein

Signal transduction through the Gal?GalNAc lectin of Entamoeba histolytica involves a spectrin-like protein

Signal transduction through the Gal-GalNAc lectin of Entamoeba histolytica involves a spectrin-like protein.

Capping followed by uroid formation in Entamoeba histolytica has been implicated in resistance against the host immune response during development of amoebiasis. The amebic actomyosin cytoskeleton is essential for such a process. A protein from the spectrin family co-localizes with the Gal-GalNAc lectin during capping of this surface protein complex. Co-localization is not observed when capping of the Gal-GalNAc lectin is specifically inhibited by production of the carboxyl-terminal region of its heavy chain that includes the lectin cytoplasmic tail. A peptide encompassing the lectin last 77 amino acids fused to glutathione-S-transferase interacts in vitro with purified spectrin. The spectrin-binding site was narrowed down to a stretch of 21 amino acids within the lectin cytoplasmic domain. This is the first report identifying an amino acid sequence involved in the interaction between the Gal-GalNAc lectin and cytoskeletal spectrin.

Identification and function analysis of spectrin-like protein in pollen tubes of lily (Lilium davidii Duch)

Identification and function analysis of spectrin-like protein in pollen tubes of lily (Lilium davidii Duch)

Golgi localization of Syne-1.

We have previously identified a Golgi-localized spectrin isoform by using an antibody to the beta-subunit of erythrocyte spectrin. In this study, we show that a screen of a lambdagt11 expression library resulted in the isolation of an approximately 5-kb partial cDNA from a Madin-Darby bovine kidney (MDBK) cell line, which encoded a polypeptide of 1697 amino acids with low, but detectable, sequence homology to spectrin (37%). A blast search revealed that this clone overlaps with the 5' end of a recently identified spectrin family member Syne-1B/Nesprin-1beta, an alternately transcribed gene with muscle-specific forms that bind acetylcholine receptor and associate with the nuclear envelope. By comparing the sequence of the MDBK clone with sequence data from the human genome database, we have determined that this cDNA represents a central portion of a very large gene ( approximately 500 kb), encoding an approximately 25-kb transcript that we refer to as Syne-1. Syne-1 encodes a large polypeptide (8406 amino acids) with multiple spectrin repeats and a region at its amino terminus with high homology to the actin binding domains of conventional spectrins. Golgi localization for this spectrin-like protein was demonstrated by expression of epitope-tagged fragments in MDBK and COS cells, identifying two distinct Golgi binding sites, and by immunofluorescence microscopy by using several different antibody preparations. One of the Golgi binding domains on Syne-1 acts as a dominant negative inhibitor that alters the structure of the Golgi complex, which collapses into a condensed structure near the centrosome in transfected epithelial cells. We conclude that the Syne-1 gene is expressed in a variety of forms that are multifunctional and are capable of functioning at both the Golgi and the nuclear envelope, perhaps linking the two organelles during muscle differentiation.

The interrelationships of actin and hyphal tip growth in the ascomycete Geotrichum candidum

Geotrichum candidum is unusual among reported hyphal ascomycetes in that its hyphae readily stain with phalloidin to reveal actin concentrated in the Spitzenkörper (SPK) and plaques associated with the plasma membrane (PM). Loss of SPK actin, but not the PM plaques, following latrunculin B treatment produces tip swelling, consistent with actin restraining tip morphology or localizing vesicle exocytosis. Tip morphogenesis may also involve a spectrin-like protein which concentrates at the apical PM in plaques unassociated with the actin plaques. Branch formation occurs with growth rates initially about 20% those of leading tips, and does not involve a morphologically detectable SPK, nor SPK-like actin ensembles, indicating the dispensibility of this structure in tip growth. Surprisingly, new tubular tips can form in the continued presence of latrunculin, consistent with alternative cellular systems, such as the spectrin-like protein, substituting for actin's critical functions.

Human spectrin Src homology 3 domain binding protein 1 regulates macropinocytosis in NIH 3T3 cells.

Macropinocytosis is an endocytic process that occurs through non-clathrin coated vesicles larger than 0.2 microm in diameter. Although macropinocytic vesicles are readily visualized in cultured cells by the introduction of fluorescent, water-soluble dyes into the culture medium, protein markers associated with this type of vesicles have not yet been well defined. Here, we report that human spectrin SH3 domain binding protein 1, or Hssh3bp1, associates with macropinosomes in NIH 3T3 fibroblasts. Hssh3bp1 macropinosomes are heterogeneous in morphology and size, do not endocytose transferrin and are resistant to brefeldin A treatment. Cytochalasin D, and wortmannin block endocytosis of fluorescent dyes into the Hssh3bp1 macropinosomes and dramatically affect their morphology. Overexpression of Hssh3bp1-green fluorescent protein abolished fusion of vesicles resulting in a decreased endocytosis of fluorescence dyes, thus suggesting a potential regulatory role of Hssh3bp1 in macropinocytosis. In the macropinosomes of NIH 3T3 cells, Hssh3bp1 associates with a 200-kDa protein that crossreacts with a monoclonal antibody to the erythroid alpha-spectrin SH3 domain. Thus macropinosomes in cells may contain a spectrin-like protein.

SPECTRIN-LIKE PROTEINS IN PLANT NUCLEI

We analysed the presence and localization of spectrin-like proteins in nuclei of various plant tissues, using several anti-erythrocyte spectrin antibodies on isolated pea nuclei and nuclei in cells. Western blots of extracted purified pea nuclei show a cross-reactive pair of bands at 220–240kDa, typical for human erythrocyte spectrin, and a prominent 60kDa band. Immunolocalization by means of confocal laser scanning microscopy reveals spectrin-like proteins in distinct spots equally distributed in the nucleoplasm and over the nuclear periphery, independent of the origin of the anti-spectrin antibodies used. In some nuclei tracks of spectrin-like proteins are also observed. No signal is present in nucleoli. The amount and intensity of signal increases when nuclei were extracted, successively, with detergents, DNase I and RNase A, and high salt, indicating that the spectrin-like protein is associated with the nuclear matrix. The labelling is similar in nuclei of various plant tissues. These data are the first that show the presence and localization of spectrin-like epitopes in plant nuclei, where they may stabilize specific interchromatin domains.

A Putative Spectrin-Containing Membrane Skeleton in Hyphal Tips of Neurospora crassa

The apical plasma membrane (PM) is important in hyphal tip growth, where it may regulate tip extensibility via its association with an appropriate membrane skeleton (MS). By cell fractionation and immunocytochemistry we show that proteins with characteristics of actin, spectrin, and integrin are associated in a MS-like manner with the PM of Neurospora crassa hyphae. The spectrin-like protein in particular is highly concentrated at the PM in the region of maximum apical expansion. This protein shares with other spectrins immunoreactivity, molecular weight, PM association, and actin binding capacity. Its distribution in hyphae suggests that it is a dominant component of the MS in true fungi and is critical to hyphal tip growth.

Immunoreactive analogues of erythrocyte ankyrin in human epidermal keratinocytes.

Using immunoblot and immunofluorescence analysis, we demonstrated the presence and localization of an immunoreactive form of erythrocyte ankyrin in human epidermal keratinocytes. Immunoblot analysis revealed that both human epidermis and cultured epidermal keratinocytes contained ankyrin-like proteins of molecular mass 210 kDa that crossreacted with antihuman erythrocyte ankyrin antibodies. Immunofluorescence microscopy revealed that the plasma membrane of epidermal keratinocytes was stained. Eccrine sweat gland cells and ductal cells were also stained. These results indicate that in human epidermal keratinocytes, eccrine sweat gland cells and ductal cells, an ankyrin-like protein is present as one of the membrane proteins. The present findings and our recent previous studies showing the presence of a spectrin-like protein (fodrin) and 4.1-like proteins in these cells enable us to suggest that a membrane skeletal protein lattice may exist in these cells.

Tyrosine phosphatase activity of lymphoma CD45 (GP180) is regulated by a direct interaction with the cytoskeleton.

GP180 is one of the major transmembrane glycoproteins in mouse T-lymphoma cells. This molecule is an isoform of CD45 and is known to contain an intrinsic protein tyrosine phosphatase (PTPase) activity. Using several complementary biochemical techniques, we have found that fodrin (a spectrin-like protein) is preferentially co-isolated with CD45 (GP180), suggesting that a complex between CD45 (GP180) and the cytoskeleton exists in mouse T-lymphoma cells. Furthermore, we have determined that this CD45 (GP180)-fodrin complex is dissociated by high salt treatment. Using in vitro binding assays, we have shown that CD45 (GP180) binds directly and specifically to fodrin (Kd approximately 1.1 nM) or spectrin (Kd approximately 3.2 nM) in a saturable manner. Additional analyses indicate that a 48-kDa phosphopeptide of CD45 (GP180) contains the fodrin/spectrin-binding domain. Most importantly, the direct binding of fodrin/spectrin to CD45 (GP180) is found to significantly stimulate the PTPase activity of CD45. Enzyme kinetic analysis indicates that fodrin and spectrin increase the Vmax of CD45 (GP180)-mediated dephosphorylation by 7.5 and 3.2-fold, respectively, without significantly changing the Km value. These results strongly suggest that the cytoskeletal proteins, fodrin and spectrin, play an important role in the regulation of the CD45 (GP180) PTPase activity during lymphocyte activation.

Identification of spectrin and calmodulin in rabbit spermiogenesis and spermatozoa

Actin was localized in the four subacrosomal bulges (Camatini et al., Eur. J. Cell Biol. 45:276-281, 1987), which characterize the sperm head of rabbit spermatozoa (Phillips, J. Ultrastruct. Res. 38:591-604, 1972), and in the postacrosomal region (Welch and O'Rand, Dev. Biol. 109:411-417, 1985; Camatini et al., Eur. J. Cell Biol. 45:276-281, 1987). Specific antibodies and indirect immunogold labelling on testis Lowicryl K4M sections and spermatozoa cryosections were used to study the distribution of calmodulin and a spectrin-like protein. This protein was also present close to the shaping membranes of the head. The results presented suggest a membrane-cytoskeletal role of actin, spectrin, and calmodulin.