Masafumi Nagayama

Temporal and spatial variations of lipid droplets during adipocyte division and differentiation

By capturing time-lapse images of primary stromal-vascular cells (SVCs) derived from rat mesenteric adipose tissue, we revealed temporal and spatial variations of lipid droplets (LDs) in individual SVCs during adipocyte differentiation. Numerous small LDs (a few micrometers in diameter) appeared in the perinuclear region at an early stage of differentiation; subsequently, several LDs grew to more than 10 μm in diameter and occupied the cytoplasm. We have developed a method for the fluorescence staining of LDs in living adipocytes. Time-lapse observation of the stained cells at higher magnification showed that nascent LDs (several 100 nm in diameter) grew into small LDs while moving from lamellipodia to the perinuclear region. We also found that adipocytes are capable of division and that they evenly distribute the LDs between two daughter cells. Immunofluorescence observations of LD-associated proteins revealed that such cell divisions of SVCs occurred even after LDs were coated with perilipin, suggesting that the “final” cell division during adipocyte differentiation occurs considerably later than that characterized in 3T3-L1 cells. Our time-lapse observations have provided a detailed account of the morphological changes that SVCs undergo during adipocyte division and differentiation.

Differential localization of non-muscle myosin II isoforms and phosphorylated regulatory light chains in human MRC-5 fibroblasts

We investigated the localization of non‐muscle myosin II isoforms and mono‐ (at serine 19) and diphosphorylated (at serine 19 and threonine 18) regulatory light chains (RLCs) in motile and non‐motile MRC‐5 fibroblasts. In migrating cells, myosin IIA localized to the lamella and throughout the posterior region. Myosin IIB colocalized with myosin IIA to the posterior region except at the very end. Diphosphorylated RLCs were detected in the restricted region where myosin IIA was enriched. In non‐motile cells, myosin IIA was enriched in peripheral stress fibers with diphosphorylated RLCs, but myosin IIB was not. Our results suggest that myosin IIA may be highly activated by diphosphorylation of RLCs and primarily involved in cell migration.

Minimum neuron density for synchronized bursts in a rat cortical culture on multi-electrode arrays

To investigate the minimum neuron and neurite densities required for synchronized bursts, we cultured rat cortical neurons on planar multi-electrode arrays (MEAs) at five plating densities (2500, 1000, 500, 250, and 100 cells/mm(2)) using two culture media: Neuron Culture Medium and Dulbeccos Modified Eagle Medium supplemented with serum (DMEM/serum). Long-term recording of spontaneous electrical activity clarified that the cultures exhibiting synchronized bursts required an initial plating density of at least 250 cells/mm(2) for Neuron Culture Medium and 500 cells/mm(2) for DMEM/serum. Immediately after electrical recording, immunocytochemistry of microtubule-associated protein 2 (MAP2) and Neurofilament 200 kD (NF200) was performed directly on MEAs to investigate the actual densities of neurons and neurites forming the networks. Immunofluorescence observation revealed that the construction of complicated neuronal networks required the same initial plating density as for synchronized bursts, and that overly sparse cultures showed significant decreases of neurons and neurites. We also found that the final densities of surviving neurons at 1 month decreased greatly compared with the initial plating densities and became saturated in denser cultures. In addition, the area of neurites and the number of nuclei were saturated in denser cultures. By comparing both the results of electrophysiological recording and immunocytochemical observation, we revealed that there is a minimum threshold of neuron densities that must be met for the exhibition of synchronized bursts. Interestingly, these minimum densities of MAP2-positive final neurons did not differ between the two culture media; the density was approximately 50 neurons/mm(2). This value was obtained in the cultures with the initial plating densities of 250 cells/mm(2) for Neuron Culture Medium and 500 cells/mm(2) for DMEM/serum.

Three-dimensional imaging of biological cells with picosecond ultrasonics

We use picosecond ultrasonics to image animal cells in vitro—a bovine aortic endothelial cell and a mouse adipose cell—fixed to Ti-coated sapphire. Tightly focused ultrashort laser pulses generate and detect GHz acoustic pulses, allowing three-dimensional imaging (x, y, and t) of the ultrasonic propagation in the cells with ∼1 μm lateral and ∼150 nm depth resolutions. Time-frequency representations of the continuous-wavelet-transform amplitude of the optical reflectivity variations inside and outside the cells show GHz Brillouin oscillations, allowing the average sound velocities of the cells and their ultrasonic attenuation to be obtained as well as the average bulk moduli.

Neuronal cell patterning on a multi-electrode array for a network analysis platform.

We studied neuronal cell patterning on a commercial multi-electrode array (MEA). We investigated the surface chemical modification of MEA in order to immobilize Poly-D-lysine (PDL) and then to pattern PDL with a photolithographic method using vacuum ultraviolet light (VUV). We have clarified that the PDL layer was not fully decomposed but was partially fragmented by short-time irradiation with VUV, resulting in a change in the cell adhesiveness of the PDL. We succeeded in patterning primary rat cortex cells without manipulating the cells on MEA more than two months. This cell-adhesiveness change induced by VUV can be applied to any immobilized PDL on other kinds of MEA and culturing substrate. We conducted electrophysiological measurements and found that the patterned neuronal cells were sufficiently matured and developed neural networks, demonstrating that our patterning method is useful for a neuronal network analysis platform.

Imaging Mechanical Properties of Living Cells by Scanning Probe Microscopy

Scanning probe microscope (SPM) has been developed as a powerful tool for obtaining high resolution topographic images of biological samples in their natural aqueous environment. SPM can also be used to evaluate mechanical properties because its probe is physically in contact with the samples during measurement. To obtain cellular stiffness with SPM, we have proposed two methods: a force modulation mode and a force mapping mode. Considering the influence of the drag force of liquids, we have successfully improved the quantitative evaluation of cellular stiffness by using the force modulation mode. Experiments performed using the two methods revealed that the local stiffness of fibroblasts was not homogeneous on the cell surface but largely varied from point to point. It was revealed that spatial and temporal distributions of cellular stiffness originate in cytoskeletal distribution, mode of cellular migration, and intracellular contractile force.

Improvement of force modulation mode with scanning probe microscopy for imaging viscoelasticity of living cells

We improved the force modulation mode with scanning probe microscopy (SPM) in order to make a quantitative evaluation of the viscoelasticity of living cells. Taking account of the viscosity of liquid medium, the vibration frequency of the cantilever was selected to be 500 Hz, and analysis of cantilever vibration was adopted for evaluation of the viscoelasticity of the samples. Consequently, we have succeeded in determining viscoelasticity distribution on living cells. The values of Youngs modulus and the coefficient of viscosity vary from 10 to 50 kPa and from 20 to 40 Pas on a cell, depending on its internal cellular structure.

Shrinking and development of lipid droplets in adipocytes during catecholamine‐induced lipolysis

Time‐lapse observation of adipocytes during catecholamine‐induced lipolysis clearly shows that shrinking of existing lipid droplets (LDs) occurs in some adipocytes and that small LDs are newly developed in almost all cells. Immunofluorescence imaging reveals that activation and localization of hormone‐sensitive lipase (HSL) on the surface of LDs, which are required for conferring maximal lipolysis, are necessary for the shrinking of the LDs. However, not all adipocytes in which phosphorylated HSL is localized on LDs exhibit shrinking of LDs. The simultaneous shrinking and development of LDs yield apparent fragmentation and dispersion of LDs in adipocytes stimulated with catecholamine.

Upside-Down Gliding of Lymnaea*

The pond snail Lymnaea stagnalis can often be observed moving upside down on its back just below the surface of the water. We have termed this form of movement “upside-down gliding.” To elucidate the mechanism of this locomotion, we performed a series of experiments involving behavioral analyses and microscopic observations. These experiments were designed (1) to measure the speed of this locomotion; (2) to determine whether the mucus secreted from the foot of Lymnaea repels water, thereby allowing the snail to exploit the surface tension of the water for upside-down gliding; and (3) to observe the beating of foot cilia in this behavior. The beating of these cilia is thought to be the primary driving force for upside-down gliding. Our results demonstrate that upside-down gliding is an efficient active process involving the secretion of mucus that floats up to the water surface to serve as a substrate upon which cilia beat to cause locomotion at the underside of the water surface.

Freezing Properties of Disaccharide Solutions: Inhibition of Hexagonal Ice Crystal Growth and Formation of Cubic Ice

Numerous studies have been undertaken to preserve living bodies and cells from freezing by addition of cryoprotective agents including natural substances (e.g., sugars and proteins) and synthetic chemicals (e.g., glycerol and dimethyl sulfoxide). Trehalose and sucrose, which consist of fructose and glucose rings connected by a glycosidic bond, are naturally occurring disaccharide compounds found in cryoprotectants. Trehalose is primarily found in animals capable of enduring cold temperatures, whereas sucrose is typically found in plants (Crowe et al., 1988). Since these molecules are too large to permeate the biomembrane, the cryoprotective mechanism is considered to be different from those of cellpermeable substances, such as glycerol and dimethyl sulfoxide. One of the considerable mechanisms of disaccharide molecules operating as a cryoprotective agent of living cells is that they protect the lipid bilayer by making the hydrogen bonding during extracellular ice formation. Based on the interactions between disaccharide molecules and the lipid bilayer, it has been suggested that the molecular mechanism underlying this cryoprotective effect is the hydrogen bonding of trehalose molecules to the bilayer head group (Sum et al., 2003). A simulation of the interaction of the lipid bilayer with trehalose has revealed that only marginal changes occur in the lipid bilayer. Another considerable mechanism of disaccharide molecules of the cryoprotective effect for living cells is that they inhibit the growth of ice crystals in extracellular space. To reveal the inhibition mechanism of disaccharides on ice crystal growth, the interaction between the disaccharide and water molecules has been investigated both by macroscopic observations (for example, Sei et al., 2001; 2002; Sei & Gonda, 2004), where the melting point of disaccharide solutions has been determined, and by microscopic observations (for example, Wang & Tominaga, 1994; Kanno & Yamazaki, 2001; Akao et al., 2001; Branca et al., 1999a) where the interaction has been investigated via Raman or infra-red spectroscopy. Sei et al. (2001; 2002) found that the melting points of trehalose and sucrose solutions were lower than those expected by the molar freezing point depression. They explained this phenomenon by considering the ratio of the hydrated water around disaccharide molecules