Patrick Chaussepied

Selective cleavage of the connector segments within the myosin-S1 heavy chain by staphylococcal protease

The existence of the two connector segments linking the tryptic 50 kDA fragment of skeletal S1 heavy chain to the adjacent 27 kDa and 20 kDa peptides was ascertained by digestion of S1 with staphylococcal protease which was found to act specifically at these particular regions. Three new peptides of M r 28 000, 48 000 and 22 000 were produced and the novel S1 derivative formed had an intact actin‐activated ATPase activity. Amino acid sequence analyses indicated that the 48 kDa and 22 kDa peptides overlap the two connector elements.

RIGOR-TYPE MUTATION IN THE KINESIN-RELATED PROTEIN HSEG5 CHANGES ITS SUBCELLULAR LOCALIZATION AND INDUCES MICROTUBULE BUNDLING

HsEg5 is a human kinesin-related motor protein essential for the formation of a bipolar mitotic spindle. It interacts with the mitotic centrosomes in a phosphorylation-dependent manner. To investigate further the mechanisms involved in targetting HsEg5 to the spindle apparatus, we expressed various mutants of HsEg5 in HeLa cells. All these mutants share a mutation of Thr-112 in the N-terminal motor domain, resulting in the inactivation of the ATP binding domain. In vitro, the HsEg5-T112N mutant motor domain showed a nucleotide-independent microtubule association, typical of a kinesin protein binding to microtubules in a rigor state. In vivo, overexpression of the HsEg5 rigor mutant in HeLa cells induced, in interphase, microtubule bundling, and, in mitosis, the formation of monopolar mitotic spindles similar to those observed after microinjection of anti-HsEg5 antibodies. Localization of the HsEg5 rigor mutant on cytoplasmic microtubules did not require the C-terminal tail domain but was lost when the stalk domain was also deleted. Sucrose gradient centrifugation experiments showed that microtubule bundling was most likely caused by the binding of HsEg5 mutants in a dimeric state. These results demonstrate that the precise subcellular localization of HsEg5 in vivo is regulated not only by the phosphorylation of the tail domain but also by the oligomeric state of the protein.

Interaction of Myosin with F-Actin: Time-Dependent Changes at the Interface Are Not Slow

The kinetics of formation of the actin-myosin complex have been reinvestigated on the minute and second time scales in sedimentation and chemical cross-linking experiments. With the sedimentation method, we found that the binding of the skeletal muscle myosin motor domain (S1) to actin filament always saturates at one S1 bound to one actin monomer (or two S1 per actin dimer), whether S1 was added slowly (17 min between additions) or rapidly (10 s between additions) to an excess of F-actin. The carbodiimide (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, EDC)-induced cross-linking of the actin-S1 complex was performed on the subsecond time scale by a new approach that combines a two-step cross-linking protocol with the rapid flow-quench technique. The results showed that the time courses of S1 cross-linking to either of the two actin monomers are identical: they are not dependent on the actin/S1 ratio in the 0.3-20-s time range. The overall data rule out a mechanism by which myosin rolls from one to the other actin monomer on the second or minute time scales. Rather, they suggest that more subtle changes occur at the actomyosin interface during the ATP cycle.

Conformational changes in actin-myosin isoforms probed by Ni(II).Gly-Gly-His reactivity.

Crucial information concerning conformational changes that occur during the mechanochemical cycle of actin–myosin complexes is lacking due to the difficulties encountered in obtaining their three-dimensional structures. To obtain such information, we employed a solution-based approach through the reaction of Ni(II)·tripeptide chelates which are able to induce protein cleavage and cross-linking reactions. Three different myosin motor domain isoforms in the presence of actin and nucleotides were treated with a library of Ni(II)·tripeptide chelates and two reactivities were observed: (1) muscle motor domains were cross-linked to actin, as also observed for the skeletal muscle isoform, while (2) the Dictyostelium discoideum motor domain was cleaved at a single locus. All Ni(II)·tripeptide chelates tested generated identical reaction products, with Ni(II)·Gly–Gly–His, containing a C-terminal carboxylate, exhibiting the highest reactivity. Mass spectrometric analysis showed that protein cleavage occurred within segment 242–265 of the Dictyostelium discoideum myosin heavy chain sequence, while the skeletal myosin cross-linking site was as localized previously within segment 506–561. Using a fusion protein consisting of the yellow and cyan variants of green fluorescent protein linked by Dictyostelium discoideum myosin segment 242–265, we demonstrated that the primary sequence of this segment alone is not a sufficient substrate for Ni(II)·Gly–Gly–His-induced cleavage. Importantly, the cross-linking and cleavage reactions both exhibited specific structural sensitivities to the nature of the nucleotide bound to the active site, validating the conformational changes suggested from crystallographic data of the actin-free myosin motor domain.

Role of charges in actomyosin interactions.

The interaction of myosin with actin is of great interest to researchers working on cell motility processes because it participates actively in the generation of actin based movement. But this interaction presents a more general interest for protein workers since its formation is a multistep binding process strongly regulated by effectors such as ATP derivatives and regulatory systems. As with most protein-protein interactions, the actomyosin complex presents a composite interface made of multiple electrostatic and hydrophobic contacts. The goal of this chapter is to identify the electrostatic contacts, to describe their role during the formation of the actomyosin interface and to discuss their contribution in the mechanism of force generation.