Spectrin alpha II and beta II isoforms interact with high affinity at the tetramerization site.

Spectrin tetramers form by the interaction of two alpha-beta dimers through two helices close to the C-terminus of a beta subunit and a single helix at the N-terminus of an alpha subunit. Early work on spectrin from solid tissues (typified by alphaII and betaII polypeptides) indicated that it forms a more stable tetramer than erythroid spectrin (alphaI-betaI). In the present study, we have probed the molecular basis of this phenomenon. We have quantified the interactions of N-terminal regions of two human alpha polypeptides (alphaI and alphaII) with the C-terminal regions of three beta isoforms (betaISigma1, betaIISigma1 and betaIISigma2). alphaII binds either betaII form with a much higher affinity than alphaI binds betaISigma1 ( K (d) values of 5-9 nM and 840 nM respectively at 25 degrees C). betaIISigma1 and betaIISigma2 are splice variants with different C-terminal extensions outside the tetramerization site: these extensions affect the rate rather than the affinity of alpha subunit interaction. alphaII spectrin interacts with each beta subunit with higher affinity than alphaI, and the betaII polypeptides have higher affinities for both alpha chains than betaISigma1. The first full repeat of the alpha subunit has a major role in determining affinity. Enthalpy changes in the alphaII-betaIISigma2 interaction are large, but the entropy change is comparatively small. The interaction is substantially reduced, but not eliminated, by concentrated salt solutions. The high affinity and slow overall kinetics of association and dissociation of alphaII-betaII spectrin may suit it well to a role in strengthening cell junctions and providing stable anchor points for transmembrane proteins at points specified by cell-adhesion molecules

Identification of human embryonic progenitor cell targeting peptides using phage display.

Human pluripotent stem (hPS) cells are capable of differentiation into derivatives of all three primary embryonic germ layers and can self-renew indefinitely. They therefore offer a potentially scalable source of replacement cells to treat a variety of degenerative diseases. The ability to reprogram adult cells to induced pluripotent stem (iPS) cells has now enabled the possibility of patient-specific hPS cells as a source of cells for disease modeling, drug discovery, and potentially, cell replacement therapies. While reprogramming technology has dramatically increased the availability of normal and diseased hPS cell lines for basic research, a major bottleneck is the critical unmet need for more efficient methods of deriving well-defined cell populations from hPS cells. Phage display is a powerful method for selecting affinity ligands that could be used for identifying and potentially purifying a variety of cell types derived from hPS cells. However, identification of specific progenitor cell-binding peptides using phage display may be hindered by the large cellular heterogeneity present in differentiating hPS cell populations. We therefore tested the hypothesis that peptides selected for their ability to bind a clonal cell line derived from hPS cells would bind early progenitor cell types emerging from differentiating hPS cells. The human embryonic stem (hES) cell-derived embryonic progenitor cell line, W10, was used and cell-targeting peptides were identified. Competition studies demonstrated specificity of peptide binding to the target cell surface. Efficient peptide targeted cell labeling was accomplished using multivalent peptide-quantum dot complexes as detected by fluorescence microscopy and flow cytometry. The cell-binding peptides were selective for differentiated hPS cells, had little or no binding on pluripotent cells, but preferential binding to certain embryonic progenitor cell lines and early endodermal hPS cell derivatives. Taken together these data suggest that selection of phage display libraries against a clonal progenitor stem cell population can be used to identify progenitor stem cell targeting peptides. The peptides may be useful for monitoring hPS cell differentiation and for the development of cell enrichment procedures to improve the efficiency of directed differentiation toward clinically relevant human cell types

A conserved sequence in calmodulin regulated spectrin-associated protein 1 links its interaction with spectrin and calmodulin to neurite outgrowth

Calmodulin regulated spectrin-associated protein 1 (CAMSAP1) is a vertebrate microtubule-binding protein, and a representative of a family of cytoskeletal proteins that arose with animals. We reported previously that the central region of the protein, which contains no recognized functional domain, inhibited neurite outgrowth when over-expressed in PC12 cells [Baines et al., Mol. Biol. Evol. 26 (2009), p. 2005]. The CKK domain (DUF1781) binds microtubules and defines the CAMSAP/ssp4 family of animal proteins (Baines et al. 2009). In the central region, three short well-conserved regions are characteristic of CAMSAP-family members. One of these, CAMSAP-conserved region 1 (CC1), bound to both ?II?1-spectrin and Ca2+/calmodulin in vitro. The binding of Ca2+/calmodulin inhibited spectrin binding. Transient expression of CC1 in PC12 cells inhibited neurite outgrowth. siRNA knockdown of CAMSAP1 inhibited neurite outgrowth in PC12 cells or primary cerebellar granule cells: this could be rescued in PC12 cells by wild-type CAMSAP1-enhanced green fluorescent protein, but not by a CC1 mutant. We conclude that CC1 represents a functional region of CAMSAP1, which links spectrin-binding to neurite outgrowth

Selectivity of Qdot peptide complexes.

<p>Embryonic progenitor cell lines were labeled with Qdot complexes in their corresponding growth media and analyzed by flow cytometry as in (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058200#pone-0058200-g005" target="_blank">Figure 5C</a>). Percentage of labeled cells was calculated by setting up gates (allowing up to 1%) using the embryonic progenitor cell line labeled with untargeted Qdots and unlabeled cells. 10,000 events were recorded for each sample. Values are from triplicate experiments and shown as mean ± standard deviation.</p

Selection of a peptide phage display library against W10 embryonic progenitor cells.

<p>(A) Peptide phages that bind to W10 embryonic progenitor cell line were enriched by 3 rounds of biopanning. PhD-12 phage display peptide library (2×10¹¹ pfu, for round 1) or amplified recovered phage (2×10¹⁰ pfu, for rounds 2 and 3) were first adsorbed against human adult dermal fibroblasts cells and then incubated with adherent W10 cells. The phages were recovered from the cell lysate and sample phage clones were sequenced. The enriched library was amplified for further rounds of selection. (B) The percentage of input phages recovered increased with each round of selection. The percentage of input phages recovered was determined by titration of plaque forming units (pfu) in the cell lysate relative to the input pfu used for each panning round. (C) Frequency and multiple sequence alignment of peptides identified as candidate peptide phage in rounds 2 and 3 of panning generated by CLUSTAL W (2.10). (D) Phylogram based on (C) denoting peptide similarities.</p

Labeling of embryonic progenitor cell line using peptide targeted Qdot605.

<p>(A) Cell targeting by fluorescent Qdots. Qdot605-ITK-SA were complexed with an excess of chemically synthesized C-terminal biotinylated peptide; unbound peptide was removed by dialysis. W10 progenitor cells were incubated for 16 h at 37°C with 5 nM of Qdot complexes, washed and imaged using a fluorescence microscope. (B) Competition with free peptide or peptide-targeted Qdots. Cells were pre-incubated with 5nM peptide, peptide targeted Qdots, or untargeted Qdots, for 30 min at 4°C, followed by addition of peptide phage (2×10¹⁰pfu) for an additional 1 h at 4°C. After washing, the recovered phage was quantified by titration. The competition is shown as percentage of no-peptide control. Values are from triplicate experiments and shown as mean ± standard deviation. Competition by corresponding free peptide or peptide-Qdot complex at 5 nM was statistically significant. Competition by uncoupled Qdots was not statistically significant (ANOVA with Dunnett’s multiple comparison tests; p values: *: <0.05. **: <0.01 and ***: <0.001) (C) Flow cytometry analysis. Cells were labeled as in (A), dissociated from the tissue culture plate using TrypLE, resuspended in PBS and analyzed in LSRFortessa flow cytometer. 10,000 events were recorded for each sample; cells were excited using the 405 nm laser and fluorescence emission was detected with the 605/12 bandpass filter. Cells labeled with W10-R3-18 peptide-Qdot complexes (green) showed higher mean fluorescent intensity than cells labeled with untargeted Qdots (red) or unlabeled W10 cells (blue).</p

Phosphorylation of a Threonine Unique to the Short C-terminal Isoform of ?II-Spectrin Links Regulation of ?-? Spectrin Interaction to Neuritogenesis

Spectrin tetramers are cytoskeletal proteins required in the formation of complex animal tissues. Mammalian alphaII- and betaII-spectrin subunits form dimers that associate head-to-head with high affinity to form tetramers, but it is not known if this interaction is regulated. We show here that the short C-terminal splice variant of betaII-spectrin (betaIIsigma2) is a substrate for phosphorylation. In vitro, protein kinase CK2 phosphorylates S2110 and T2159; PKA phosphorylates T2159. Anti-phospho-T2159 peptide antibody detected phosphorylated betaIIsigma2 in Cos-1 cells. Immunoreactivity was increased in Cos-1 cells by treatment with forskolin, indicating that phosphorylation is promoted by elevated cAMP. The effect of forskolin was counteracted by the cAMP-dependent kinase inhibitor, H89. In vitro, PKA phosphorylation of an active fragment of betaIIsigma2 greatly reduced its interaction with alphaII-spectrin at the tetramerization site. Mutation of T2159 to alanine eliminated inhibition by phosphorylation. Among the processes that require spectrin in mammals is the formation of neurites (incipient nerve axons). We tested the relationship of spectrin phosphorylation to neuritogenesis by transfecting the neuronal cell line, PC12, with enhanced green fluorescent protein-coupled fragments of betaIIsigma2-spectrin predicted to act as inhibitors of spectrin tetramer formation. Both wild type and T2159E mutant fragments allowed neuritogenesis in PC12 cells in response to nerve growth factor. The mutant T2159A inhibited neuritogenesis. Since the T2159A mutant represents a high affinity inhibitor of tetramer formation, we conclude that tetramers are requisite for neuritogenesis. Furthermore, since both the T2159E mutant and the wild-type allow neuritogenesis, we conclude that the short C-terminal betaII-spectrin is phosphorylated during this process