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Yasushi Sako

Publications and source records attributed to Yasushi Sako.

13 recordsLinked to original sources
PubMed2006

Epidermal growth factor-regulated activation of Rac GTPase enhances CD44 cleavage by metalloproteinase disintegrin ADAM10.

Invasive tumour cells, such as gliomas, frequently express EGF (epidermal growth factor) receptor at a high level and they exhibit enhanced cell migration in response to EGF. We reported previously that tumour cell migration is associated with ectodomain cleavage of CD44, the major adhesion molecule that is implicated in tumour invasion and metastasis, and that the cleavage is enhanced by ligation of CD44. In the present study, we show that EGF promotes CD44 cleavage and CD44-dependent cell migration. Introduction of a dominant-negative mutant of the small GTPase Rac1 or depletion of Rac1 by RNAi (RNA interference) abrogated CD44 cleavage induced by EGF. Treatment with PD98059, an inhibitor for MEK (mitogen-activated protein kinase/extracellular-signal-regulated kinase kinase), also suppressed the CD44 cleavage. Furthermore, RNAi studies showed that EGF induced ADAM10 (a disintegrin and metalloproteinase 10)-dependent CD44 cleavage and cell migration. These results indicate that EGF induces ADAM10-mediated CD44 cleavage through Rac1 and mitogen-activated protein kinase activation, and thereby promotes tumour cell migration and invasion.

ADAM Proteins↗
PubMed2006

Covalent immobilization of epidermal growth factor molecules for single-molecule imaging analysis of intracellular signaling.

We have developed cell stimulative system by covalently immobilized signalling molecules on the surface of coverslips on which cells are later cultured. N-(6-maleimidocaproyloxy)sulfo-succinimide (sulfo-EMCS) cross-links the amino-terminal of epidermal growth factors (EGF) with the thiol-modified glass surface without degrading EGF's physiological activity. The glass surface was covered up to about 1.0 EGF moleculesnm(-2) with uniform density. This density can be controlled by changing concentration of the maleimide-modified EGF in the solution reacting with the thiol-modified glass coverslips. When the density of EGF was only slightly lower than that of EGF receptor dimers, cellular response was dramatically decreased. The EGF receptor molecules bound with the immobilized EGF were prevented from lateral diffusion and internalization and kept their initial position. These properties are useful for quantitative, spatial and temporal control of the input signal stimulating cells in cellular signaling system studies. In addition, the immobility of the EGF in this system makes suitable targets for stable single-molecule observation under total internal reflection fluorescence microscopy (TIR-FM) to study EGF signalling mechanism, preventing lateral diffusion and internalization of EGF receptors. Here we show results of single-molecule observations of the association and dissociation between phosphorylated EGF receptors and Cy3-labeled growth factor receptor-binding protein 2 (Grb2) proteins in A431 cells stimulated by the immobilized EGF and discuss the utility of this method for the study of intracellular signal transduction.

Biological Assay↗
PubMed2006

Formation of signal transduction complexes during immobile phase of NGFR movements.

Nerve growth factor (NGF) is a secreted neurotrophin involved in the differentiation, growth, and maintenance of neurons. Here, we have used single-molecule imaging to characterize the behavior of Cy3-tagged NGF after binding to receptor complexes on the surfaces of PC12 cells. We show that NGF-receptor complexes have two distinct diffusive states, characterized as mobile and immobile phase. The transition between the two diffusive states occurred reversibly with duration times determined by a single rate limiting process. The abrupt transition to the immobile phase often occurred simultaneously with the clustering of NGF-receptor complexes. Immobilization depended on the phosphorylation of the TrkA NGF-receptor. Using dual-color imaging, it was demonstrated that the membrane recruitment of the intercellular signaling protein occurs with NGF-receptor complexes in the immobile phase indicating signal transduction occurs during this phase. Thus, NGF signaling is performed through a repetitive random process to induce transient formation of signaling complexes.

Animals↗
PubMed2005

Single-molecule analysis of epidermal growth factor signaling that leads to ultrasensitive calcium response.

Quantitative relationships between inputs and outputs of signaling systems are fundamental information for the understanding of the mechanism of signal transduction. Here we report the correlation between the number of epidermal growth factor (EGF) bindings and the response probability of intracellular calcium elevation. Binding of EGF molecules and changes of intracellular calcium concentration were measured for identical HeLa human epithelial cells. It was found that 300 molecules of EGF were enough to induce calcium response in half of the cells. This number is quite small compared to the number of EGF receptors (EGFR) expressed on the cell surface (50,000). There was a sigmoidal correlation between the response probability and the number of EGF bindings, meaning an ultrasensitive reaction. Analysis of the cluster size distribution of EGF demonstrated that dimerization of EGFR contributes to this switch-like ultrasensitive response. Single-molecule analysis revealed that EGF bound faster to clusters of EGFR than to monomers. This property should be important for effective formation of signaling dimers of EGFR under very small numbers of EGF bindings and suggests that the expression of excess amounts of EGFR on the cell surface is required to prepare predimers of EGFR with a large association rate constant to EGF.

Animals↗
PubMed2005

Trafficking of a ligand-receptor complex on the growth cones as an essential step for the uptake of nerve growth factor at the distal end of the axon: a single-molecule analysis.

The behavior of single molecules of neurotrophins on growth cones was observed by the use of the fluorescent conjugate of nerve growth factor (NGF), Cy3-NGF. After the application of 0.4 nm Cy3-NGF, chick dorsal root ganglion growth cones responded within 1 min of adding the stimulus by expanding their lamellipodia. Only 40 molecules of Cy3-NGF, which occupied <5% of the estimated total binding sites on a single growth cone, were required to initiate the motile responses. After binding to the high-affinity receptor, Cy3-NGF displayed lateral diffusion on the membrane of the growth cones with a diffusion constant of 0.3 microm2 s(-1). The behavior of Cy3-NGF was shifted to a one-directional rearward movement toward the central region of the growth cone. The one-directional movement of Cy3-NGF displayed the same rate as the rearward flow of actin, approximately 4 microm/min. This movement could be stopped by the application of the potent inhibitor of actin polymerization, latrunculin B. Molecules of Cy3-NGF were suggested to be internalized in the vicinity of the central region of the growth cone during this rearward trafficking, because Cy3-NGF remained in the growth cone after the growth cones had been exposed to an acidic surrounding medium: acidic medium causes the complete dissociation of Cy3-NGF from the receptors on the surface of growth cones. These results suggested that actin-driven trafficking of the NGF receptor complex is an essential step for the accumulation and endocytosis of NGF at the growth cone and for the retrograde transport of NGF toward the cell body.

Animals↗
PubMed2004

EGF signalling amplification induced by dynamic clustering of EGFR.

Lateral interaction is an important feature of various types of cell surface receptors including the receptor tyrosine kinases (RTKs). Here we report that dynamic lateral interaction produces amplification and variation in signalling of the EGF receptor, a member of RTKs. Binding of EGF is known to induce transphosphorylation inside EGFR dimers. Using single-molecule techniques, the relationship between EGF binding and EGFR phosphorylation has been determined. The number of phosphorylated EGFR molecules became larger than that of EGF binding as unliganded EGFR was phosphorylated, meaning an amplification of EGF signalling. EGFR formed clusters continuously exchanging their elements through thermal diffusion, and direct and/or indirect lateral interactions. As a result, various types of activation sites differing in number of activated receptors were generated. Amplification required no cytoplasmic factors and was observed on semi-intact cells for a wide range of number of EGFR molecules (10(4)-10(6) per cell) suggesting generality of this process.

Adenosine Triphosphate↗
PubMed2003

Engagement of CD44 promotes Rac activation and CD44 cleavage during tumor cell migration.

CD44 is a major cell surface adhesion molecule for hyaluronan, a component of the extracellular matrix, and is implicated in tumor metastasis and invasion. We reported previously that hyaluronan oligosaccharides induce CD44 cleavage from tumor cells. Here we show that engagement of CD44 promotes CD44 cleavage and tumor cell migration, both of which were suppressed by a metalloproteinase inhibitor KB-R7785 and tissue inhibitor of metalloproteinases-1 (TIMP-1) but not by TIMP-2. We also present evidence that blockade of metalloproteinase-disintegrin ADAM10 (a disintegrin and metalloproteinase 10) by RNA interference suppresses CD44 cleavage induced by its ligation. Engagement of CD44 concurrently induced activation of the small GTPase Rac1 and led to drastic changes in cell morphology and actin cytoskeleton with redistribution of CD44 to newly generated membrane ruffling areas. A fluorescence resonance energy transfer approach to visualize GTP-bound Rac1 in living cells revealed the localization of the active Rac1 in the leading edge of the membrane ruffling areas upon ligation of CD44. Taken together, our results indicate that the cleavage of CD44 catalyzed by ADAM10 is augmented by the intracellular signaling elicited by engagement of CD44, through Rac-mediated cytoskeletal rearrangement, and suggest that CD44 cleavage contributes to the migration and invasion of tumor cells.

ADAM Proteins↗
PubMed2003

Optical bioimaging: from living tissue to a single molecule: single-molecule visualization of cell signaling processes of epidermal growth factor receptor.

Single-molecule imaging is an ideal technology to study molecular mechanisms of biological reactions in vitro. Recently, this technology has been extended to real-time observation of fluorescent dye-labeled molecules in living cells. Total internal reflection fluorescence microscopy is the major technique for this purpose. Using this technique, we have studied the process of early signal transduction of epidermal growth factor (EGF) in single molecules: binding of EGF to its receptor (EGFR) on the cell surface, dimerization of EGFR induced by binding of EGF, fluctuation of the structure of EGFR clusters, activation of EGFR through tyrosine phosphorylations on its cytoplasmic domain, and recognition of activated EGFR by a cytoplasmic adaptor protein, Grb2. EGF induces intracellular calcium response, sometimes caused by less than one hundred EGF molecules. Single-molecule studies suggested that this highly sensitive response to EGF was due to the amplification of the EGFR signal using dynamic clustering, reorganization of the dimers, and lateral mobility of EGFR on the cell surface. Through these studies, single-molecule analysis has proven to be a powerful technology to analyze intracellular protein systems.

Animals↗
PubMed2003

Single-molecule visualization in cell biology.

Recent progress in single-molecule detection techniques has allowed us to visualize the dynamic behaviour and reaction kinetics of individual biological molecules inside living cells. Single-molecule visualization provides a direct way to quantify, with a high spatial and temporal resolution, biological events inside cells at the single-molecule level. In this article, we discuss how single-molecule visualization can be used in cell biology.

Animals↗
PubMed2002

Total internal reflection fluorescence microscopy for single-molecule imaging in living cells.

Marvelous background rejection in total internal reflection fluorescence microscopy (TIR-FM) has made it possible to visualize single-fluorophores in living cells. Cell signaling proteins including peptide hormones, membrane receptors, small G proteins, cytoplasmic kinases as well as small signaling compounds have been conjugated with single chemical fluorophore or tagged with green fluorescent proteins and visualized in living cells. In this review, the reasons why single-molecule analysis is essential for studies of intracellular protein systems such as cell signaling system are discussed, the instrumentation of TIR-FM for single-molecule imaging in living cells is explained, and how single molecule visualization has been used in cell biology is illustrated by way of two examples: signaling of epidermal growth factor in mammalian cells and chemotaxis of Dictyostelium amoeba along a cAMP gradient. Single-molecule analysis is an ideal method to quantify the parameters of reaction dynamics and kinetics of unitary processes within intracellular protein systems. Knowledge of these parameters is crucial for the understanding of the molecular mechanisms underlying intracellular events, thus single-molecule imaging in living cells will be one of the major technologies in cellular nanobiology.

Animals↗