Jochen D'Haese

Regulatory light chains of myosin from the obliquely-striated body wall muscle of Lumbricus terrestris

The myosin-linked Ca2+-regulatory system of invertebrate muscle has been described in detail only for some molluscan muscles. In this muscle-type a regulatory light chain which is readily removed by EDTA was found to serve as the regulatory component [ 1,2]. So far, only for the myosin from scallop adductor [2,3] and clam foot muscles [4] such regulatory light chains have been found, whereas in other molluscan muscles a similar system could not be demonstrated [5,6]. In contrast to these molluscan muscles the majority of invertebrate muscles are doubly regulated by a myosinand an actin-linked regulatory system [5,7]. Here the Ca2+-regulation of obliquely striated muscle was analyzed with particular attention to the mode of regulation confered by the myosin component. It is shown that, as in molluscan muscles the myosin of obliquely striated muscle posseses a regulatory light chain that is extractable with EDTA. As with molluscan musclesthe light chain may be reversibly removed and alsotifunctionally replaced by the P light chain of vertebrate smooth muscle.

In vivo and in vitro effects of imidacloprid on sheep keds (Melophagus ovinus): a light and electron microscopic study

Abstract The effects of imidacloprid (Advantage) on sheep keds (Melophagus ovinus Linné 1758) were studied in vivo and in vitro by means of direct observation (monitored on video tape) and by light and electron microscopy. It was found that:1. Imidacloprid acted rapidly on all motile stages of the sheep keds. Within 3–4 min after exposure they became immobile and their legs and the abdomen started tetanic trembling movements for 15–30 min, leading to death.2. The compound is apparently taken up by the body, since it also acted on those sheep keds that had been exclusively exposed to imidacloprid-contaminated filter papers.3. The compound is available and active for more than 1 month in the wool of sheep; even rainfall does not reduce its efficacy. Body contact between treated mother sheep and their lambs protects them from infestation with these ectoparasites.4. The compound initiates an ultimately lethal destruction of the ganglia, nerve chords and related muscle fibers, as can be seen in electron micrographs.

Purification and functional characterization of an 85-kDa gelsolin from the ascidians Microcosmus sulcatus and Phallusia mammilata.

From the pharyngeal baskets of the ascidians Microcosmus sulcatus and Phallusia mammilata we have purified an 85-kDa protein that is characterized as a member of the gelsolin family. These proteins from both species show the same behaviour in functional assays. The ascidian gelsolin binds two actin monomers in a highly cooperative manner. This complex formation is Ca(2+)-dependent, but not completely reversible, as on removal of Ca2+ one actin monomer dissociates leaving a 1:1 complex between gelsolin and G-actin. The properties of F-actin severing and G-actin nucleation depend on the presence of free Ca2+ in a micromolar range, with half maximum activation at about 3 x 10(-6) M. The protein becomes inactivated when Ca2+ concentrations of 0.5 mM are exceeded. Fragmentation of F-actin by the ascidian gelsolin is comparably fast to that of vertebrate gelsolin. A steady state of actin fragmentation is reached within 2-4 s. Promotion of G-actin nucleation is also comparable to that of vertebrate gelsolin. Regarding functional aspects, the ascidian gelsolin is more closely related to vertebrate gelsolin than to an arthropod gelsolin from crayfish tail muscle.

C-terminally deleted fragments of 40-kDa earthworm actin modulator still show gelsolin activities

C‐ and N‐terminally truncated fragments of earthworm gelsolin were constructed, cloned and expressed in Escherichia coli. G‐actin‐binding properties of these fragments and their influences on the polymeric state of actin were investigated. A construct lacking a large part of the third segment [E(1–295)] supports actin nucleation similar to the complete protein and shows reduced actin fragmentation property, but is no longer Ca2+‐sensitive in its activity. The first and the second segments (E1 and E2) each contain one actin‐binding site. In contrast to human gelsolin, E1 in combination with a short N‐terminal region of E2 is not sufficient for the F‐actin‐severing activity of the protein.

EF-Hand Proteins and the Regulation of Actin-Myosin Interaction in the Eutardigrade Hypsibius klebelsbergi (Tardigrada)

Many tardigrade species resist harsh environmental conditions by entering anhydrobiosis or cryobiosis. Desiccation as well as freeze resistance probably leads to changes of the ionic balance that includes the intracellular calcium concentration. In order to search for protein modifications affecting the calcium homoeostasis, we studied the regulatory system controlling actin-myosin interaction of the eutardigrade Hypsibius klebelsbergi and identified full-length cDNA clones for troponin C (TnC, 824 bp), calmodulin (CaM, 1,407 bp), essential myosin light chain (eMLC, 1,015 bp), and regulatory myosin light chain (rMLC, 984 bp) from a cDNA library. All four proteins belong to the EF-hand superfamily typified by a calcium coordinating helix-loop-helix motif. Further, we cloned and obtained recombinant TnC and both MLCs. CaM and TnC revealed four and two potential calcium-binding domains, respectively. Gel mobility shift assays demonstrated calcium-induced conformational transition of TnC. From both MLCs, only the rMLC showed one potential N-terminal EF-hand domain. Additionally, sequence properties suggest phosphorylation of this myosin light chain. Based on our results, we suggest a dual-regulated system at least in somatic muscles for tardigrades with a calcium-dependent tropomyosin-troponin complex bound to the actin filaments and a phosphorylation of the rMLC turning on and off both actin and myosin. Our results indicate no special modifications of the molecular structure and function of the EF-hand proteins in tardigrades. Phylogenetic trees of 131 TnCs, 96 rMLCs, and 62 eMLCs indicate affinities to Ecdysozoa, but also to some other taxa suggesting that our results reflect the complex evolution of these proteins rather than phylogenetic relationships.

Four paralog gelsolin genes are differentially expressed in the earthworm Lumbricus terrestris

We have identified and characterized four distinct variants of the gelsolin-related protein (EWAM P1-P4) in the earthworm L. terrestris. All of these proteins biochemically qualify as gelsolins since they sever actin filaments in a calcium dependent manner. P1, P2 and P3 are present in the Lumbricus body wall muscle whereas in the gizzard muscle P3 and P4 were found. P1-P4 are encoded by four paralog genes and are differentially expressed in various muscle cell tissues. While the genes for P1 and P2 contain one intron, there was no intron in both P3 and P4 genes. The coding sequences consist of 1104bp (368 amino acids) for P1/P4 and 1101bp (367 amino acids) for P2/P3. Corresponding genes were confirmed by northern blot analysis which revealed three (calculated lengths: 3100, 2300 and 2100 nucleotides) and two (calculated lengths: 2300 and 1700 nucleotides) mRNA transcripts in the body wall and the gizzard, respectively. EWAM mRNA was localized by fluorescence in situ hybridization in the body wall and the gizzard muscle. P1 mRNA was detected in the inner proximal layers of both the circular and longitudinal muscle of the body wall whereas in the gizzard no significant staining was observed for P1. P2-P4 mRNAs were abundant in the outer distal layers of both the circular and the longitudinal muscles of both body wall and gizzard. The differential expression of four paralog gelsolin genes suggests a functional adaptation of different muscle cells with respect to actin filament turnover and modulation of its polymer state.

Gelsolin in Onychophora and Tardigrada with notes on its variability in the Ecdysozoa.

Rearrangements of the filamentous actin network involve a broad range of actin binding proteins. Among these, the gelsolin proteins sever actin filaments, cap their fast growing end and nucleate actin assembly in a calcium-dependent manner. Here, we focus on the gelsolin of the onychophoran Peripatoides novaezealandiae and the eutardigrade Hypsibius dujardini. From the cDNA of P. novaezealandiae we obtained the complete coding sequence with an open reading frame of 2178bp. It encodes a protein of 726 amino acids with a calculated molecular mass of 82,610.9Da and a pI of 5.57. This sequence is comprised of six segments (S1-S6). However, analysis of data from TardiBase reveals that the gelsolin of the eutardigrade Hypsibius dujardini has only three segments (S1-S3). The coding sequence consist of 1119bp for 373 amino acids with a calculated molecular mass of 42,440.95Da and a pI of 6.17. The Peripatoides and Hypsibius gelsolin revealed both conserved binding motifs for G-actin, F-actin and phosphatidylinositol 4,5-bisphosphate (PIP2), along with a full set of type-1 and type-2 Ca2+-binding sites which could result in the binding of eight and four calcium ions, respectively. Both gelsolin proteins lack a C-terminal latch-helix indicating a more rapid activation in the submicromolar Ca2+ range. We suggest that a gelsolin with three segments was present in the last common ancestor of the ecdysozoan clade Panarthropoda (Onychophora, Tardigrada, Arthropoda), primarily because the gelsolin of all non-Ecdysozoa studied so far (except Chordata) reveals this number of segments. Mapping of our molecular data onto a well-established phylogeny revealed that the number of gelsolin segments does not correlate with the phylogenetic lineage but rather with particular functional demands to alter the kinetics of actin polymerization.