Kazutoshi Gohara

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.

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.

DYNAMICAL SYSTEMS EXCITED BY TEMPORAL INPUTS: FRACTAL TRANSITION BETWEEN EXCITED ATTRACTORS

This paper presents a framework for dissipative dynamical systems excited by external temporal inputs. We introduce a set {Il} of temporal inputs with finite intervals. The set {Il} defines two other sets of dynamical systems. The first is the set of continuous dynamical systems that are defined by a set {fl} of vector fields on the hyper-cylindrical phase space ℳ. The second is the set of discrete dynamical systems that are defined by a set {gl} of iterated functions on the global Poincare section Σ. When the inputs are switched stochastically, a trajectory in the space ℳ converges to an attractive invariant set with fractal-like structure. We can analytically prove this result when all of the iterated functions satisfy a contraction property. Even without this property, we can numerically show that an attractive invariant set with fractal-like structure exists.

Continuous hitting movements modeled from the perspective of dynamical systems with temporal input

The kinematics of continuously repeated forehand and backhand tennis strokes were measured and analyzed in terms of a dynamical system with temporal input. The tennis strokes were performed under two conditions: when the same input pattern was repeated and when two different input patterns were switched stochastically. The condition with the two periodic inputs revealed that there were two different trajectories in cylindrical space, while the condition with switching input induced transitions between these two trajectories, which were considered as excited attractors. The transitions between the two excited attractors were characterized by a fractal-like feature that could be described by a simple Cantor set with rotation. This result suggests that continuous hitting movements have a hierarchical fractal structure, as was expected from the theory of dynamical systems with external temporal input.

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.

Diffraction microscopy using 20 kV electron beam for multiwall carbon nanotubes

Diffraction microscopy with iterative phase retrieval using a 20kV electron beam was carried out to explore the possibility of high-resolution imaging for radiation-sensitive materials. Fine, homogeneous, and isolated multiwall carbon nanotubes (MWCNTs) were used as specimens. To avoid lens aberrations, the diffraction patterns were recorded without a postspecimen lens. One- and two-dimensional iterative phase retrievals were executed. Images reconstructed from the diffraction pattern alone showed a characteristic structure of MWCNTs with the finest feature corresponding to a carbon wall spacing of 0.34nm.

10-kV diffractive imaging using newly developed electron diffraction microscope.

A new electron diffraction microscope based on a conventional scanning electron microscope (SEM), for obtaining atomic-level resolution images without causing serious damage to the specimen, has been developed. This microscope in the relatively low-voltage region makes it possible to observe specimens at suitable resolution and record diffraction patterns. Using the microscope we accomplished 10-kV diffractive imaging with the iterative phase retrieval and reconstructed the structure of a multi-wall carbon nanotube with its finest feature corresponding to 0.34-nm carbon wall spacing. These results demonstrate the possibility of seamless connection between observing specimens by SEM and obtaining their images at high resolution by diffractive imaging.

FRACTAL TRANSITION: HIERARCHICAL STRUCTURE AND NOISE EFFECT

A Sierpinski gasket with continuous trajectories is presented as an example of the fractal transition that characterizes the behavior of dissipative dynamical systems excited by external temporal inputs. Using this example, we investigate the fractal transition from two points of views, i.e. a hierarchical structure and a noise effect. Depending on internal and external parameters, the structure can be geometrically classified as one of three types, i.e. totally disconnected, just-touching, and overlapping. For the totally disconnected structure, continuous trajectories and their starting points can be characterized by a definite hierarchical tree structure. Even for the just-touching and overlapping structure, a similar hierarchy exists. White noise contaminating the external inputs breaks the hierarchy. In particular, small clustered structures are sensitive to the noise. In such a case, the difference between trajectories and starting points is remarkable in the hierarchy.

Dissociation of the insulin receptor from caveolae during TNFα‐induced insulin resistance and its recovery by d‐PDMP

Previously, we demonstrated that an inhibitor of ganglioside biosynthesis, d‐PDMP, could restore impaired insulin signaling in tumor necrosis factor α (TNFα)‐treated adipocytes by blocking the increase of GM3 ganglioside. Here, we analyzed the interaction between insulin receptor (IR) and GM3 in the plasma membranes using immunoelectron microscopy. In normal adipocytes, most GM3 molecules localized at planar and non‐caveolar regions. Approximately 19% of IR molecules were detected in caveolar regions. The relative ratio of IRs associated with caveolae in TNFα‐treated adipocytes was decreased to one‐fifth of that in normal adipocytes, but this decrease was restored by d‐PDMP. Thus, we could obtain direct evidence that insulin resistance is a membrane microdomain disorder caused by aberrant expression of ganglioside.

Cell Patterning Using a Template of Microstructured Organosilane Layer Fabricated by Vacuum Ultraviolet Light Lithography

Micropatterning techniques have become increasingly important in cellular biology. Cell patterning is achieved by various methods. Photolithography is one of the most popular methods, and several light sources (e.g., excimer lasers and mercury lamps) are used for that purpose. Vacuum ultraviolet (VUV) light that can be produced by an excimer lamp is advantageous for fabricating material patterns, since it can decompose organic materials directly and efficiently without photoresist or photosensitive materials. Despite the advantages, applications of VUV light to pattern biological materials are few. We have investigated cell patterning by using a template of a microstructured organosilane layer fabricated by VUV lithography. We first made a template of a microstructured organosilane layer by VUV lithography. Cell adhesive materials (poly(d-lysine) and polyethyleneimine) were chemically immobilized on the organosilane template, producing a cell adhesive material pattern. Primary rat cardiac and neuronal cells were successfully patterned by culturing them on the pattern substrate. Long-term culturing was attained for up to two weeks for cardiac cells and two months for cortex cells. We have discussed the reproducibility of cell patterning and made suggestions to improve it.