Single-molecule observation of protein-protein interactions in the chaperonin system.

We have analyzed the dynamics of the chaperonin (GroEL)-cochaperonin (GroES) interaction at the single-molecule level. In the presence of ATP and non-native protein, binding of GroES to the immobilized GroEL occurred at a rate that is consistent with bulk kinetics measurements. However, the release of GroES from GroEL occurred after a lag period ( approximately 3 s) that was not recognized in earlier bulk-phase studies. This observation suggests a new kinetic intermediate in the GroEL-GroES reaction pathway.

Imaging of single fluorescent molecules using video-rate confocal microscopy.

Single fluorescent molecules in aqueous solution were imaged for the first time at video-rate using Nipkow disk-type confocal microscopy. Performance of this method was evaluated by imaging single kinesin molecules labeled with fluorescent dyes of tetramethylrhodamine (TMR) or IC5. Photodecomposition lifetimes of the fluorophores were approximately 10 s for TMR and approximately 2 s for IC5 under the incident laser power of 0.5 W/mm(2). Both the fluorescence intensity and the photobleaching rate were proportional to the laser power from 0.65 to 3 W/mm(2). 2D sliding movement of single kinesin molecules along microtubules on glass surface and 3D Brownian motion of individual kinesin molecules in viscous solution could be observed using this microscopy. These results indicated that this method could be applicable to the study of single molecular events in living cells at real time.

Characterization of single actomyosin rigor bonds: load dependence of lifetime and mechanical properties.

Load dependence of the lifetime of the rigor bonds formed between a single myosin molecule (either heavy meromyosin, HMM, or myosin subfragment-1, S1) and actin filament was examined in the absence of nucleotide by pulling the barbed end of the actin filament with optical tweezers. For S1, the relationship between the lifetime (tau) and the externally imposed load (F) at absolute temperature T could be expressed as tau(F) = tau(0).exp(-F.d/k(B)T) with tau(0) of 67 s and an apparent interaction distance d of 2.4 nm (k(B) is the Boltzmann constant). The relationship for HMM was expressed by the sum of two exponentials, with two sets of tau(0) and d being, respectively, 62 s and 2.7 nm, and 950 s and 1.4 nm. The fast component of HMM coincides with tau(F) for S1, suggesting that the fast component corresponds to single-headed binding and the slow component to double-headed binding. These large interaction distances, which may be a common characteristic of motor proteins, are attributed to the geometry for applying an external load. The pulling experiment has also allowed direct estimation of the number of myosin molecules interacting with an actin filament. Actin filaments tethered to a single HMM molecule underwent extensive rotational Brownian motion, indicating a low torsional stiffness for HMM. From these results, we discuss the characteristics of interaction between actin and myosin, with the focus on the manner of binding of myosin.

Association of immunolocalization of matrix metalloproteinase 1 with ovulation in hCG-treated rabbit ovary.

Matrix metalloproteinase 1, MMP-1, which was previously called interstitial collagenase, is necessary for extracellular matrix reconstruction. The immunolocalization of the latent form of MMP-1 (proMMP-1) was examined in the ovulatory process in hCG-treated (100 iu per animal) rabbit ovaries. Immunoreactive products of proMMP-1 were identified by the avidin-biotin peroxidase complex formation using an anti-rabbit proMMP-1 polyclonal antibody. ProMMP-1 was distributed in the cytoplasm of theca interna cells, theca externa cells, interstitial glands and germinal epithelium throughout ovulation. However, at 9 or 10 h after hCG treatment, this enzyme was identified in several capillary lumina around the apex of preovulatory follicles. In addition, the staining density of immunoreactive products apparently increased in granulosa cells and theca interna cells around the orifice of the ruptured follicle 10 h after the stimulation. These results indicate that the spatiotemporal appearance of proMMP-1 in ovulation may be closely associated with the initiation of rupture of the follicle.