Chika Okimura

Electroporation of adherent cells with low sample volumes on a microscope stage

SUMMARY The labeling of specific molecules and their artificial control in living cells are powerful techniques for investigating intracellular molecular dynamics. To use these techniques, molecular compounds (hereinafter described simply as ‘samples’) need to be loaded into cells. Electroporation techniques are exploited to load membrane-impermeant samples into cells. Here, we developed a new electroporator with four special characteristics. (1) Electric pulses are applied to the adherent cells directly, without removing them from the substratum. (2) Samples can be loaded into the adherent cells while observing them on the stage of an inverted microscope. (3) Only 2 μl of sample solution is sufficient. (4) The device is very easy to use, as the cuvette, which is connected to the tip of a commercially available auto-pipette, is manipulated by hand. Using our device, we loaded a fluorescent probe of actin filaments, Alexa Fluor 546 phalloidin, into migrating keratocytes. The level of this probe in the cells could be easily adjusted by changing its concentration in the electroporation medium. Samples could be loaded into keratocytes, neutrophil-like HL-60 cells and Dictyostelium cells on a coverslip, and keratocytes on an elastic silicone substratum. The new device should be useful for a wide range of adherent cells and allow electroporation for cells on various types of the substrata.

Myosin-II-Mediated Directional Migration of Dictyostelium Cells in Response to Cyclic Stretching of Substratum

Living cells are constantly subjected to various mechanical stimulations, such as shear flow, osmotic pressure, and hardness of substratum. They must sense the mechanical aspects of their environment and respond appropriately for proper cell function. Cells adhering to substrata must receive and respond to mechanical stimuli from the substrata to decide their shape and/or migrating direction. In response to cyclic stretching of the elastic substratum, intracellular stress fibers in fibroblasts and endothelial, osteosarcoma, and smooth muscle cells are rearranged perpendicular to the stretching direction, and the shape of those cells becomes extended in this new direction. In the case of migrating Dictyostelium cells, cyclic stretching regulates the direction of migration, and not the shape, of the cell. The cells migrate in a direction perpendicular to that of the stretching. However, the molecular mechanisms that induce the directional migration remain unknown. Here, using a microstretching device, we recorded green fluorescent protein (GFP)-myosin-II dynamics in Dictyostelium cells on an elastic substratum under cyclic stretching. Repeated stretching induced myosin II localization equally on both stretching sides in the cells. Although myosin-II-null cells migrated randomly, myosin-II-null cells expressing a variant of myosin II that cannot hydrolyze ATP migrated perpendicular to the stretching. These results indicate that Dictyostelium cells accumulate myosin II at the portion of the cell where a large strain is received and migrate in a direction other than that of the portion where myosin II accumulated. This polarity generation for migration does not require the contraction of actomyosin.

The Role of Stress Fibers in the Shape Determination Mechanism of Fish Keratocytes.

Crawling cells have characteristic shapes that are a function of their cell types. How their different shapes are determined is an interesting question. Fish epithelial keratocytes are an ideal material for investigating cell shape determination, because they maintain a nearly constant fan shape during their crawling locomotion. We compared the shape and related molecular mechanisms in keratocytes from different fish species to elucidate the key mechanisms that determine cell shape. Wide keratocytes from cichlids applied large traction forces at the rear due to large focal adhesions, and showed a spatially loose gradient associated with actin retrograde flow rate, whereas round keratocytes from black tetra applied low traction forces at the rear small focal adhesions and showed a spatially steep gradient of actin retrograde flow rate. Laser ablation of stress fibers (contractile fibers connected to rear focal adhesions) in wide keratocytes from cichlids increased the actin retrograde flow rate and led to slowed leading-edge extension near the ablated region. Thus, stress fibers might play an important role in the mechanism of maintaining cell shape by regulating the actin retrograde flow rate.

Shape and Area of Keratocytes Are Related to the Distribution and Magnitude of Their Traction Forces.

Fish epidermal keratocytes maintain an overall fan shape during their crawling migration. The shape-determination mechanism has been described theoretically and experimentally on the basis of graded radial extension of the leading edge, but the relationship between shape and traction forces has not been clarified. Migrating keratocytes can be divided into fragments by treatment with the protein kinase inhibitor staurosporine. Fragments containing a nucleus and cytoplasm behave as mini-keratocytes and maintain the same fan shape as the original cells. We measured the shape of the leading edge, together with the areas of the ventral region and traction forces, of keratocytes and mini-keratocytes. The shapes of keratocytes and mini-keratocytes were similar. Mini-keratocytes exerted traction forces at the rear left and right ends, just like keratocytes. The magnitude of the traction forces was proportional to the area of the keratocytes and mini-keratocytes. The myosin II ATPase inhibitor blebbistatin decreased the forces at the rear left and right ends of the keratocytes and expanded their shape laterally. These results suggest that keratocyte shape depends on the distribution of the traction forces, and that the magnitude of the traction forces depends on the area of the cells.

Fast-crawling cell types migrate to avoid the direction of periodic substratum stretching.

ABSTRACT To investigate the relationship between mechanical stimuli from substrata and related cell functions, one of the most useful techniques is the application of mechanical stimuli via periodic stretching of elastic substrata. In response to this stimulus, Dictyostelium discoideum cells migrate in a direction perpendicular to the stretching direction. The origins of directional migration, higher migration velocity in the direction perpendicular to the stretching direction or the higher probability of a switch of migration direction to perpendicular to the stretching direction, however, remain unknown. In this study, we applied periodic stretching stimuli to neutrophil-like differentiated HL-60 cells, which migrate perpendicular to the direction of stretch. Detailed analysis of the trajectories of HL-60 cells and Dictyostelium cells obtained in a previous study revealed that the higher probability of a switch of migration direction to that perpendicular to the direction of stretching was the main cause of such directional migration. This directional migration appears to be a strategy adopted by fast-crawling cells in which they do not migrate faster in the direction they want to go, but migrate to avoid a direction they do not want to go.

Hybrid mechanosensing system to generate the polarity needed for migration in fish keratocytes

ABSTRACT Crawling cells can generate polarity for migration in response to forces applied from the substratum. Such reaction varies according to cell type: there are both fast- and slow-crawling cells. In response to periodic stretching of the elastic substratum, the intracellular stress fibers in slow-crawling cells, such as fibroblasts, rearrange themselves perpendicular to the direction of stretching, with the result that the shape of the cells extends in that direction; whereas fast-crawling cells, such as neutrophil-like differentiated HL-60 cells and Dictyostelium cells, which have no stress fibers, migrate perpendicular to the stretching direction. Fish epidermal keratocytes are another type of fast-crawling cell. However, they have stress fibers in the cell body, which gives them a typical slow-crawling cell structure. In response to periodic stretching of the elastic substratum, intact keratocytes rearrange their stress fibers perpendicular to the direction of stretching in the same way as fibroblasts and migrate parallel to the stretching direction, while blebbistatin-treated stress fiber-less keratocytes migrate perpendicular to the stretching direction, in the same way as seen in HL-60 cells and Dictyostelium cells. Our results indicate that keratocytes have a hybrid mechanosensing system that comprises elements of both fast- and slow-crawling cells, to generate the polarity needed for migration.

The molecular dynamics of crawling migration in microtubule-disrupted keratocytes

Cell-crawling migration plays an essential role in complex biological phenomena. It is now generally believed that many processes essential to such migration are regulated by microtubules in many cells, including fibroblasts and neurons. However, keratocytes treated with nocodazole, which is an inhibitor of microtubule polymerization – and even keratocyte fragments that contain no microtubules – migrate at the same velocity and with the same directionality as normal keratocytes. In this study, we discovered that not only these migration properties, but also the molecular dynamics that regulate such properties, such as the retrograde flow rate of actin filaments, distributions of vinculin and myosin II, and traction forces, are also the same in nocodazole-treated keratocytes as those in untreated keratocytes. These results suggest that microtubules are not in fact required for crawling migration of keratocytes, either in terms of migrating properties or of intracellular molecular dynamics.

Rotation of stress fibers as a single wheel in migrating fish keratocytes

Crawling migration plays an essential role in a variety of biological phenomena, including development, wound healing, and immune system function. Keratocytes are wound-healing cells in fish skin. Expansion of the leading edge of keratocytes and retraction of the rear are respectively induced by actin polymerization and contraction of stress fibers in the same way as for other cell types. Interestingly, stress fibers in keratocytes align almost perpendicular to the migration-direction. It seems that in order to efficiently retract the rear, it is better that the stress fibers align parallel to it. From the unique alignment of stress fibers in keratocytes, we speculated that the stress fibers may play a role for migration other than the retraction. Here, we reveal that the stress fibers are stereoscopically arranged so as to surround the cytoplasm in the cell body; we directly show, in sequential three-dimensional recordings, their rolling motion during migration. Removal of the stress fibers decreased migration velocity and induced the collapse of the left-right balance of crawling migration. The rotation of these stress fibers plays the role of a “wheel” in crawling migration of keratocytes.