Multiple-color fluorescence imaging of chromosomes and microtubules in living cells.

Microscopic observation of fluorescently-stained intracellular molecules within a living cell provides a straightforward approach to understanding their temporal and spatial relationships. However, exposure to the excitation light used to visualize these fluorescently-stained molecules can be toxic to the cells. Here we describe several important considerations in microscope instrumentation and experimental conditions for avoiding the toxicity associated with observing living fluorescently-stained cells.

Dynamic interaction between BAF and emerin revealed by FRAP, FLIP, and FRET analyses in living HeLa cells.

Barrier-to-autointegration factor (BAF) is a conserved 10 kDa DNA-binding protein. BAF interacts with LEM-domain proteins including emerin, LAP2 beta, and MAN1 in the inner nuclear membrane. Using fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP), we compared the mobility of BAF to its partners emerin, LAP2 beta, and MAN1 in living HeLa cells.

Dynamics of homologous chromosome pairing during meiotic prophase in fission yeast.

Pairing of homologous chromosomes is important for homologous recombination and correct chromosome segregation during meiosis. It has been proposed that telomere clustering, nuclear oscillation, and recombination during meiotic prophase facilitate homologous chromosome pairing in fission yeast. Here we examined the contributions of these chromosomal events to homologous chromosome pairing, by directly observing the dynamics of chromosomal loci in living cells of fission yeast.

Neural mechanisms of spatial stimulus-response compatibility: the effect of crossed-hand position.

Previous psychological experiments have indicated the existence of a visual-proprioceptive interaction in spatial stimulus-response compatibility (SSRC) tasks, but there is little specific information on the neural basis of such interaction in humans. Using functional magnetic resonance imaging (fMRI), we compared the neural activity associated with two different aspects of spatial coding: the coding of the "internal" spatial position of motor-response effectors (i.e.

Asymmetric activation to the context-dependent right in the right inferior frontal region.

In human spatial recognition, right and left are not recognized symmetrically. Although there have been many studies on the hemispheric asymmetry of the human brain, asymmetries in high-level recognition (such as independence from input or output hemisphere) have not been studied extensively. We found that the human brain recognizes right and left asymmetrically in high-level recognition.

Emerin binding to Btf, a death-promoting transcriptional repressor, is disrupted by a missense mutation that causes Emery-Dreifuss muscular dystrophy.

Loss of functional emerin, a nuclear membrane protein, causes X-linked recessive Emery-Dreifuss muscular dystrophy. In a yeast two-hybrid screen, we found that emerin interacts with Btf, a death-promoting transcriptional repressor, which is expressed at high levels in skeletal muscle. Biochemical analysis showed that emerin binds Btf with an equilibrium affinity (KD) of 100 nm.

Chemomechanical coupling in F1-ATPase revealed by simultaneous observation of nucleotide kinetics and rotation.

F(1)-ATPase is a rotary molecular motor in which unidirectional rotation of the central gamma subunit is powered by ATP hydrolysis in three catalytic sites arranged 120 degrees apart around gamma. To study how hydrolysis reactions produce mechanical rotation, we observed rotation under an optical microscope to see which of the three sites bound and released a fluorescent ATP analog. Assuming that the analog mimics authentic ATP, the following scheme emerges: (i) in the ATP-waiting state, one site, dictated by the orientation of gamma, is empty, whereas the other two bind a nucleotide; (ii) ATP binding to the empty site drives an approximately 80 degrees rotation of gamma; (iii) this triggers a reaction(s), hydrolysis and/or phosphate release, but not ADP release in the site that bound ATP one step earlier; (iv) completion of this reaction induces further approximately 40 degrees rotation.

Regulation of oxygen transport during brain activation: stimulus-induced hemodynamic responses in human and animal cortices.

BACKGROUND: The correlation between regional changes in neuronal activity and changes in hemodynamics is a major issue for noninvasive neuroimaging techniques such as functional magnetic resonance imaging (fMRI) and near-infrared optical imaging (NIOI). A tight coupling of these changes has been assumed to elucidate brain function from data obtained with those techniques. In the present study, we investigated the relationship between neuronal activity and hemodynamic responses in the occipital cortex of humans during visual stimulation and in the somatosensory cortex of rats during peripheral nerve stimulation.

Cytoplasmic dynein in fungi: insights from nuclear migration.

Cytoplasmic dynein is a microtubule motor that mediates various biological processes, including nuclear migration and organelle transport, by moving on microtubules while associated with various cellular structures. The association of dynein with cellular structures and the activation of its motility are crucial steps in dynein-dependent processes. However, the mechanisms involved remain largely unknown.

Human precentral cortical activation patterns during saccade tasks: an fMRI comparison with activation during intentional eyeblink tasks.

Some fMRI studies in humans have located the frontal eye field (FEF) in two distinct regions along the precentral sulcus (PCS): one localized more medically, in the superior precentral sulcus (supPCR) at the junction with the superior frontal sulcus, and the other localized more laterally, along the medial part of the inferior precentral sulcus (infPCR). However, there has been no evidence of any different task activations between the regions. In the present study using fMRI, we have compared activation patterns in the regions near the PCS during saccade, fixation, and eyeblink tasks.

Discrete stochastic process underlying perceptual rivalry.

In perceptual rivalry such as ambiguous figure perception and binocular rivalry, the conscious percept spontaneously alternates between two stable interpretations of an unchanging stimulus. It is well known that the time intervals of the perceptual alternation follow a gamma distribution (GD), but its implication for the alternation mechanism has not been clarified. We examined quantitatively GDs fitted to alternation intervals, and found that the shape-determining parameter alpha of the GDs took natural numbers.

Cell cycle behavior of human HP1 subtypes: distinct molecular domains of HP1 are required for their centromeric localization during interphase and metaphase.

Heterochromatin protein 1 (HP1) plays an important role in heterochromatin formation. Three subtypes of HP1, namely HP1alpha, beta, and gamma, have been identified in humans. In this study, using yellow fluorescent protein (YFP) fusion constructs, we examined the intracellular localization of human HP1 subtypes during the cell cycle.

Monopolar spindle attachment of sister chromatids is ensured by two distinct mechanisms at the first meiotic division in fission yeast.

At meiosis I, sister chromatids attach to the same spindle pole (i.e. monopolar attachment).

Functional MRI of brain activation evoked by intentional eye blinking.

Eye blinking is not only a reflexive action to protect the ocular surface from injury and desiccation; it can also be done intentionally. However, only a few studies have investigated the brain mechanism controlling intentional blinking, and there are still inconsistencies among the reported activation patterns in the human brain evoked by intentional blinking. In monkeys, some areas where blinking is evoked by electrical microstimulation have been found in the premotor areas and in the posterior parietal cortex.

Linear element formation and their role in meiotic sister chromatid cohesion and chromosome pairing.

Fission yeast does not form synaptonemal complexes in meiotic prophase. Instead, linear elements appear that resemble the axial cores of other eukaryotes. They have been proposed to be minimal structures necessary for proper meiotic chromosome functions.

Higher plant myosin XI moves processively on actin with 35 nm steps at high velocity.

High velocity cytoplasmic streaming is found in various plant cells from algae to angiosperms. We characterized mechanical and enzymatic properties of a higher plant myosin purified from tobacco bright yellow-2 cells, responsible for cytoplasmic streaming, having a 175 kDa heavy chain and calmodulin light chains. Sequence analysis shows it to be a class XI myosin and a dimer with six IQ motifs in the light chain-binding domains of each heavy chain.

Multispectral imaging fluorescence microscopy for living cells.

Multispectral imaging technologies have been widely used in fields of astronomy and remote sensing. Interdisciplinary approaches developed in, for example, the National Aeronautics and Space Administration (NASA, USA), the Jet Propulsion Laboratory (JPL, USA), or the Communications Research Laboratory (CRL, Japan) have extended the application areas of these technologies from planetary systems to cellular systems. Here we overview multispectral imaging systems that have been devised for microscope applications.

Live cell imaging: approaches for studying protein dynamics in living cells.

In the last decade, the long-standing biologist's dream of seeing the molecular events within the living cell came true. This technological achievement is largely due to the development of fluorescence microscopy technologies and the advent of green fluorescent protein as a fluorescent probe. Such imaging technologies allowed us to determine the subcellular localization, mobility and transport pathways of specific proteins and even visualize protein-protein interactions of single molecules in living cells.

An electronic read-out circuit for time-domain terahertz imaging with photoconductive antennas.

We have developed a signal amplifier for the detection of weak repetitive pulsed signals superimposed on a large background. It is based on a double gated integrator principle with two channels locked to a sequence of alternate signal and background pulses. The circuit is compact and cost effective, and thus suitable for use in large numbers with detector arrays, for example in terahertz imaging.

Dorsal visual cortex activity elicited by posture change in a visuo-tactile matching task.

To investigate the process of crossmodal spatial recognition, we examined the effect of posture change on the recognition of a tactile stimulus position. The task was to judge whether a visual and a tactile stimulus, presented to the left or right, were on the same or different sides while subjects crossed or uncrossed their hands. Under a condition which removed the effect of response bias to the left and right, the dorsal visual cortex (area 18/19) and the precuneus were more activated in the crossed hands condition.

Spectral imaging fluorescence microscopy.

The spectral resolution of fluorescence microscope images in living cells is achieved by using a confocal laser scanning microscope equipped with grating optics. This capability of temporal and spectral resolution is especially useful for detecting spectral changes of a fluorescent dye; for example, those associated with fluorescence resonance energy transfer (FRET). Using the spectral imaging fluorescence microscope system, it is also possible to resolve emitted signals from fluorescent dyes that have spectra largely overlapping with each other, such as fluorescein isothiocyanate (FITC) and green fluorescent protein (GFP).

Mechanical Properties of a Single-Headed Processive Motor, Inner-Arm Dynein Subspecies-c of ChlamydomonasStudied at the Single Molecule Level.

Dynein from inner arms of Chlamydomonasflagella contains sevendistinct subspecies, a through g. Several lines of evidence suggest thesesubspecies play important roles in generating flagellar beating and thatthe different subspecies are functionally diverse. To evaluate theirroles and diversity, the mechanical properties of subspecies-c, which isa single-headed motor, were examined using optical trap nanometry.

BAF is required for emerin assembly into the reforming nuclear envelope.

Mutations in emerin cause the X-linked recessive form of Emery-Dreifuss muscular dystrophy (EDMD). Emerin localizes at the inner membrane of the nuclear envelope (NE) during interphase, and diffuses into the ER when the NE disassembles during mitosis. We analyzed the recruitment of wildtype and mutant GFP-tagged emerin proteins during nuclear envelope assembly in living HeLa cells.

Dynamic behavior of microtubules during dynein-dependent nuclear migrations of meiotic prophase in fission yeast.

During meiotic prophase in fission yeast, the nucleus migrates back and forth between the two ends of the cell, led by the spindle pole body (SPB). This nuclear oscillation is dependent on astral microtubules radiating from the SPB and a microtubule motor, cytoplasmic dynein. Here we have examined the dynamic behavior of astral microtubules labeled with the green fluorescent protein during meiotic prophase with the use of optical sectioning microscopy.