Kazuyo Ohashi

Short-term retention of actin filament binding proteins on lamellipodial actin bundles

Actin filaments are organised into sub‐compartments of meshwork and bundles in lamellipodia. Localisation of fascin, the LIM and SH3 domain protein 1 (lasp‐1), and lasp‐2 to the bundles suggest their involvement in that organisation; however, their contributions remain unclear. We have compared the turnover of these proteins with actin at the bundle. After photobleaching, EGFP‐actin recovered inwards from the bundle tip, consistent with the retrograde flow by treadmilling. In contrast, the recovery of EGFP‐fascin, ‐lasp‐1 and ‐lasp‐2 occurred from the anterograde direction. These results suggest that these molecules would participate in the stabilisation of bundles but not in initiation.

Fourier transform infrared spectroscopic analysis of the intact zona pellucida of the mammalian egg : Changes in the secondary structure of bovine zona pellucida proteins during fertilization

The zona pellucida is the acellular transparent envelope surrounding the mammalian oocyte. An analysis of the changes in the structures of zona pellucida proteins is essential for understanding the molecular mechanisms underlying the important physiological roles of the zona during fertilization and prelmplantatlon. The hardening of the zona caused by the structural changes during fertilization is generally accepted to be responsible for blocking polyspermy. In this study, we analyzed changes in the secondary structure of the zona during fertilization by Fourier transform infrared (FTIR) spectroscopy and transmission electron microscopy. The predominance of β-sheet structure in porcine ovarian egg zona proteins in water was ascertained using FTIR spectra. α-Helix structure was also present. The attenuated total reflection (ATR)-FTIR spectrum of intact, unsolubilized porcine zonae pellucidae from ovarian eggs Indicated that the zona proteins in the native zona pellucida also have β-structure as the main constituent. Attenuated total reflectlon-FTIR spectroscopy of intact bovine zona pellucida obtained from ovarian and fertilized eggs at the blastocyst stage revealed that the β-structure content Increased during fertilization. Furthermore, a reduction of the thickness of the zona during fertilization was observed using transmission electron microscopy. Therefore, the change in the zona architecture that causes hardening of the zona during fertilization is accompanied by changes in the secondary structure of the zona proteins.

Contribution of the LIM Domain and Nebulin-Repeats to the Interaction of Lasp-2 with Actin Filaments and Focal Adhesions

Lasp-2 binds to actin filaments and concentrates in the actin bundles of filopodia and lamellipodia in neural cells and focal adhesions in fibroblastic cells. Lasp-2 has three structural regions: a LIM domain, a nebulin-repeat region, and an SH3 domain; however, the region(s) responsible for its interactions with actin filaments and focal adhesions are still unclear. In this study, we revealed that the N-terminal fragment from the LIM domain to the first nebulin-repeat module (LIM-n1) retained actin-binding activity and showed a similar subcellular localization to full-length lasp-2 in neural cells. The LIM domain fragment did not interact with actin filaments or localize to actin filament bundles. In contrast, LIM-n1 showed a clear subcellular localization to filopodial actin bundles. Although truncation of the LIM domain caused the loss of F-actin binding activity and the accumulation of filopodial actin bundles, these truncated fragments localized to focal adhesions. These results suggest that lasp-2 interactions with actin filaments are mediated through the cooperation of the LIM domain and the first nebulin-repeat module in vitro and in vivo. Actin filament binding activity may be a major contributor to the subcellular localization of lasp-2 to filopodia but is not crucial for lasp-2 recruitment to focal adhesions.

The Role of Actin-Binding Protein Filamin A in Cellular Stiffness and Morphology Studied by Wide-Range Scanning Probe Microscopy

The role of filamin A (FLNa) in the organization of stress fibers has been studied by comparing the mechanical properties of FLNa-deficient human melanoma cells (M2 cells) and M2 sub-line expressing FLNa (A7 cells). We measured both the topographies and the elasticity distributions of M2 and A7 cells by using a wide-range scanning probe microscopy. In A7 cells, we observed aligned fibrous structures, whereas in M2 cells, fibrous structures were dispersed randomly. Immunofluorescent observation revealed that the aligned fibrous structures in A7 cells were stress fibers generating intracellular tension. On the other hand, the reticular structures observed in M2 cells did not correspond to actin filaments. The cellular stiffness of A7 cells was approximately twice than that of M2 cells, indicating that A7 cells produce larger contractile force through the stress fibers. These results suggest that FLNa stabilizes the stress fibers and increases the cellular stiffness.

Mechanical Response of Single Filamin A (ABP‐280) Molecules and Its Role in the Actin/Filamin A Gel

Actin/filamin A gel plays important roles in mechanical response of cells. We found a force (50 to 220 pN)‐induced unfolding of single filamin A molecules using AFM, and have proposed a hypothesis on the role of single filamin A in the novel property of viscoelasticity of actin/filamin A gel. We also investigated structure and its dynamics of actin/filamin A gel formed in a giant liposome using fluorescence microscopy.

I-protein forms cage-like aggregates of myosin in vitro.

I-protein was mixed with myosin before or after myosin filaments were reconstituted. In both cases, I-protein seemed to accelerate the myosin assembly. The binding of I-protein to myosin filaments was tested by sedimentation experiments and SDS-polyacrylamide gel electrophoresis. In a low ionic strength solution at pH 6.5, the binding ratio of I-protein to myosin was 1:40 by molar ratio when the I-protein molecules highly specifically bound to myosin filaments. I-protein could maximally bind to myosin filaments at the molar ratio of 1:2.7. In this case, excess I-protein molecules remained in the supernatant after sedimentation, although the unbound I-protein could still bind to myosin filaments. Electron microscopic observations revealed that I-protein bundled myosin filaments in the low ionic strength solution (pH 6.5). Cage-like structures which were very similar to the Mg-paracrystals of non-muscle myosins were formed at pH 7.2. In gel filtration, the apparent molecular mass of I-protein was 100 kDa, while it was 50 kDa in SDS gel electrophoresis. Therefore, I-protein is regarded to be a homodimer of a 50 kDa subunit and can divalently bind to myosin molecules.