Novel Modification of a Confirmatory SMA Sequencing Assay that Can Be Used to Determine SMN2 Copy Number.

Promising treatments for spinal muscular atrophy (SMA), the leading genetic cause of infant mortality, prompted calls for inclusion in newborn screening (NBS). In January 2018, the New England Newborn Screening Program (NENSP) began statewide screening for SMA using a tiered algorithm looking for the absence of SMN1 Exon 7. When results from the first and second tier needed reconciliation, we developed and validated a third tier DNA sequencing assay to ensure the presence or absence of SMN1 Exon 7. All nine infants referred to specialty centers through NBS showed single base substitution of c.840C>T, and were confirmed to have SMA. Further, a minor sequencing protocol modification allowed the estimation of SMN2 copy number in SMA affected patients; we developed and validated a copy-number assay yielding 100% match with seven previously characterized specimens of SMA patients. All nine SMA-affected infants found through NBS were also assayed for SMN2 copy number. Results were comparable but not 100% matched with those that were reported by independent diagnostic laboratories. In conclusion, a sequencing protocol confirms NBS findings from real-time qPCR, and its modified application allows NBS programs that have sequencing capabilities to provide SMN2 copy numbers without the need for additional instrumentation.

Ablation of smooth muscle caldesmon affects the relaxation kinetics of arterial muscle.

Smooth muscle caldesmon (h-CaD) is an actin- and myosin-binding protein that reversibly inhibits the actomyosin ATPase activity in vitro. To test the function of h-CaD in vivo, we eliminated its expression in mice. The h-CaD-null animals appeared normal and fertile, although the litter size was smaller. Tissues from the homozygotes lacked h-CaD and exhibited upregulation of the non-muscle isoform, l-CaD, in visceral, but not vascular tonic smooth muscles. While the Ca(2+) sensitivity of force generation of h-CaD-deficient smooth muscle remained largely unchanged, the kinetic behavior during relaxation in arteries was different. Both intact and permeabilized arterial smooth muscle tissues from the knockout animals relaxed more slowly than those of the wild type. Since this difference occurred after myosin dephosphorylation was complete, the kinetic effect most likely resulted from slower detachment of unphosphorylated crossbridges. Detailed analyses revealed that the apparently slower relaxation of h-CaD-null smooth muscle was due to an increase in the amplitude of a slower component of the biphasic tension decay. While the identity of this slower process has not been unequivocally determined, we propose it reflects a thin filament state that elicits fewer re-attached crossbridges. Our finding that h-CaD modulates the rate of smooth muscle relaxation clearly supports a role in the control of vascular tone.

Erk1/2 MAPK and caldesmon differentially regulate podosome dynamics in A7r5 vascular smooth muscle cells.

We tested the hypothesis that the MEK/Erk/caldesmon phosphorylation cascade regulates PKC-mediated podosome dynamics in A7r5 cells. We observed the phosphorylation of MEK, Erk and caldesmon, and their translocation to the podosomes upon phorbol dibutyrate (PDBu) stimulation, together with the nuclear translocation of phospho-MEK and phospho-Erk. After MEK inhibition by U0126, Erk translocated to the interconnected actin-rich columns but failed to translocate to the nucleus, suggesting that podosomes served as a site for Erk phosphorylation. The interconnected actin-rich columns in U0126-treated, PDBu-stimulated cells contained alpha-actinin, caldesmon, vinculin, and metalloproteinase-2. Caldesmon and vinculin became integrated with F-actin at the columns, in contrast to their typical location at the ring of podosomes. Live-imaging experiments suggested the growth of these columns from podosomes that were slow to disassemble. The observed modulation of podosome size and life time in A7r5 cells overexpressing wild-type and phosphorylation-deficient caldesmon-GFP mutants in comparison to untransfected cells suggests that caldesmon and caldesmon phosphorylation modulate podosome dynamics in A7r5 cells. These results suggest that Erk1/2 and caldesmon differentially modulate PKC-mediated formation and/or dynamics of podosomes in A7r5 vascular smooth muscle cells.

Direct interaction between caldesmon and cortactin.

Actin polymerization and depolymerization plays a central role in controlling a wide spectrum of cellular processes. There are many actin-binding proteins in eukaryotic cells. Their roles in the remodeling of the actin architecture and whether they work cooperatively await further study. Caldesmon (CaD) is an actin-binding protein present in nearly all mammalian cells. Cortactin is another actin-binding protein found mainly in the cell cortex. There have been no reports suggesting that CaD and cortactin interact with each other or work as partners. Here, we present evidence that CaD binds cortactin directly by overlay, pull-down assays, ELISA, and by column chromatography. The interaction involves the N-terminal region of cortactin and the C-terminal region of CaD, and appears to be enhanced by divalent metal ions. Cortactin competes with both full-length CaD and its C-terminal fragment for actin binding. Binding of cortactin partially alleviates the inhibitory effect of CaD on the actomyosin ATPase activity. Not only can binding be demonstrated in vitro, the two proteins also co-localize in activated cells at the cortex. Whether such interactions bear any functional significance awaits further investigation.

Phosphorylation of caldesmon during smooth muscle contraction and cell migration or proliferation.

The actin-binding protein caldesmon (CaD) exists both in smooth muscle (the heavy isoform, h-CaD) and non-muscle cells (the light isoform, l-CaD). In smooth muscles h-CaD binds to myosin and actin simultaneously and modulates the actomyosin interaction. In non-muscle cells l-CaD binds to actin and stabilizes the actin stress fibers; it may also mediate the interaction between actin and non-muscle myosins. Both h- and l-CaD are phosphorylated in vivo upon stimulation. The major phosphorylation sites of h-CaD when activated by phorbol ester are the Erk-specific sites, modification of which is attenuated by the MEK inhibitor PD98059. The same sites in l-CaD are also phosphorylated when cells are stimulated to migrate, whereas in dividing cells l-CaD is phosphorylated more extensively, presumably by cdc2 kinase. Both Erk and cdc2 are members of the MAPK family. Thus it appears that CaD is a downstream effector of the Ras signaling pathways. Significantly, the phosphorylatable serine residues shared by both CaD isoforms are in the C-terminal region that also contains the actin-binding sites. Biochemical and structural studies indicated that phosphorylation of CaD at the Erk sites is accompanied by a conformational change that partially dissociates CaD from actin. Such a structural change in h-CaD exposes the myosin-binding sites on the actin surface and allows actomyosin interactions in smooth muscles. In the case of non-muscle cells, the change in l-CaD weakens the stability of the actin filament and facilitates its disassembly. Indeed, the level of l-CaD modification correlates very well in a reciprocal manner with the level of actin stress fibers. Since both cell migration and cell division require dynamic remodeling of actin cytoskeleton that leads to cell shape changes, phosphorylation of CaD may therefore serve as a plausible means to regulate these processes. Thus CaD not only links the smooth muscle contractility and non-muscle motility, but also provides a common mechanism for the regulation of cell migration and cell proliferation.

Phosphorylated l-caldesmon is involved in disassembly of actin stress fibers and postmitotic spreading.

The function of the ubiquitous actin-binding protein, caldesmon (l-CaD) in mammalian non-muscle cells remains elusive. During mitosis, l-CaD becomes markedly phosphorylated at Ser497 and Ser527 (in the rat sequence), therefore, it has been suggested that l-CaD is involved in cytokinesis by inhibiting the actomyosin interaction until it is phosphorylated, although direct in vivo evidence is still missing. In the present study, we used F-actin staining and specific antibodies against these two phosphorylation sites of l-CaD to simultaneously monitor actin assembly and l-CaD phosphorylation. Our observations demonstrated that the level of l-CaD phosphorylation undergoes dynamic changes during the cell cycle. The spatial and temporal distributions of phospho-CaD do not correlate with cytokinesis per se, but rather, with the level of actin bundles in a reciprocal manner. The highest l-CaD phosphorylation level coincides with the disassembly of actin cytoskeleton during mitotic cell rounding. Ser-to-Ala mutations at these two positions prevent stress fibers from disassembly upon migratory stimulation. In addition, phospho-CaD appears to colocalize with nascent focal adhesion complexes during postmitotic spreading. These findings suggest that l-CaD phosphorylation plays an important role not only in cytoskeleton remodeling during cell shape changes, but also in cell spreading and migration.

Crystal structures of S100A6 in the Ca(2+)-free and Ca(2+)-bound states: the calcium sensor mechanism of S100 proteins revealed at atomic resolution.

S100A6 is a member of the S100 family of Ca(2+) binding proteins, which have come to play an important role in the diagnosis of cancer due to their overexpression in various tumor cells. We have determined the crystal structures of human S100A6 in the Ca(2+)-free and Ca(2+)-bound states to resolutions of 1.15 A and 1.44 A, respectively. Ca(2+) binding is responsible for a dramatic change in the global shape and charge distribution of the S100A6 dimer, leading to the exposure of two symmetrically positioned target binding sites. The results are consistent with S100A6, and most likely other S100 proteins, functioning as Ca(2+) sensors in a way analogous to the prototypical sensors calmodulin and troponin C. The structures have important implications for our understanding of target binding and cooperativity of Ca(2+) binding in the S100 family.