Reversible excitation light-induced enhancement of fluorescence of live mammalian mitochondria.

During continuous irradiation with near-ultraviolet light (l = 36510 nm; 16 mW/mm(2)) for 2-3 min, live mammalian cells increased reversibly the intensity of one or more peaks of their autofluorescence spectrum from an initial ('ground') level to a two- to threefold higher ('active') level. The effect is characterized by the existence of two states of quantum efficiency and a mechanism of transition that expresses a threshold and a refractory period. It appears that mitochondria are the principal sources of the rising autofluorescence intensity; however, not all mitochondria are capable of expressing it. Studying cells from various organisms that belong to various branches of the phylogenetic tree, we found the rapid increase of autofluorescence only in placental mammalian cells. We speculate that the effect may point to the ability of placental mammalian mitochondria to generate pulsating light signals.

Altered drug resistance of microtubules in cells exposed to infrared light pulses: are microtubules the "nerves" of cells?

This article describes the first quantitative assay of the response of an entire population of cultured mammalian cells to a pulsating near-infrared signal. The assay measures the change of resistance to nocodazole of reconstituted cytoplasmic asters of irradiated cells. Using this assay on CV1 cells, I obtained results suggesting that pulsating near-infrared signals of 1 s pulse length reduced the stability of the radial microtubules around the centrosome. The results are consistent with the interpretation that the centrosome responded to the light by sending signals along its radial array of microtubules whose stability was then altered. The results may be an example of a more general function of the centrosome to integrate exogenous signals and send response signals along microtubules to various sites within the cell.

Autofluorescence of live purple bacteria in the near infrared.

We have developed a novel microscope with which to study the fluorescence of cells in the near-infrared region (lambda = 750-2500 nm). For one of its first applications we report on the autofluorescence of live purple bacteria, Rhodospirillum rubrum, and suggest that the autofluorescent component is bacteriochlorophyll. The rapid fading of the autofluorescence of fixed bacteria and of purified bacteriochlorophyll suggests that the live bacteria are able to regenerate their pigment with a time constant of approximately 20 s.

Changes of cell behavior by near-infrared signals.

3T3 mouse fibroblasts responded differently to specific near-infrared signals from epithelial CV1 cells. Furthermore, signals with the same wavelength and energy changed the percentages of attracted and repelled 3T3 cells if their intensity modulation was altered. I found this result in a 22 month long study which established a spectrum of motile responses of 781 individual 3T3 cells and 148 CV1 cells to the near-infrared emissions of microscopic, pulsating light sources using the infrared spot-irradiation phase-contrast (IRSIP) microscope [Albrecht-Buehler, 1991: J. Cell Biol. 114:493-502]. Thus the response of cultured, mammalian cells to near-infrared light signals is not merely a matter of total energy absorption by certain cytoplasmic components. Since it seems to depend on the cell type and the temporal pattern in which the light energy is emitted, it appears to imply the existence of a new kind of cellular information.

Cellular infrared detector appears to be contained in the centrosome.

Previous experiments have suggested that 3T3 cells were able to extend pseudopodia toward latex particles up to 60 microns away from the cell body if the particles were irradiated by an infrared beam in the range of 700-900 nm [Albrecht-Buehler, 1991: J. Cell Biol. 114:493-502]. The present article reports that this response of cells to infrared light can be inhibited if the cell center is simultaneously irradiated with a beam of the same light. In marked contrast, the cells responded normally to the presence of infrared light scattering particles if the second beam irradiated other parts of the cell body. The results imply that the cellular mechanism of infrared detection is located at the cell center. The infrared sensing mechanism remains intact in enucleated cells and in cells which were incubated in monensin to vesiculate their Golgi apparatus and inhibit their Golgi functions. Accordingly, it is proposed that the centrosome which contains the centrioles is the only remaining candidate in the cell center for a cellular detection device for the direction of infrared signal sources. The results support an earlier suggestion that centrioles may be such detection devices [Albrecht-Buehler, 1981: Cell Motil. Cytoskeleton 1:237-245].

The simulation of microgravity conditions on the ground.

This chapter defines weightlessness as the condition where the acceleration of an object is independent of its mass. Applying this definition to the clinostat, it argues that the clinostat is very limited as a simulator of microgravity because it (a) generates centrifugal forces, (b) generates particle oscillations with mass-dependent amplitudes of speed and phase shifts relative to the clinorotation, (c) is unable to remove globally the scalar effects of gravity such as hydrostatic pressure, which are independent of the direction of gravity in the first place, and, (d) generates more convective mixing of the gaseous or liquid environment of the test object, rather than eliminating it, as would true weightlessness. It is proposed that attempts to simulate microgravity must accept the simulation of one aspect of microgravity at a time, and urges that the suppression of convective currents be a major feature of experimental methods that simulate microgravity.

Rudimentary form of cellular "vision".

BHK cells were inoculated sparsely on one face ("sparse- or s-face") of a thin glass film whose opposite face was covered with a 2- to 3-day-old confluent layer of BHK cells ("confluent- or c-face"). After 7 hr of attaching and spreading in the absence of visible light, most of the cells on the s-face traversed with their long axes the direction of the whorls of the confluent cells on the c-face directly opposed. The effect was inhibited by a thin metal coating of the glass films. The results suggest that the cells were able to detect the orientation of others by signals that penetrated glass but not thin metallic films and, therefore, appeared to be carried by electromagnetic radiation. In contrast, the effect was not influenced by a thin coat of silicone on the glass, suggesting that the wavelength of this radiation is likely to be in the red to infrared range. The ability of cells to detect the direction of others by electromagnetic signals points to a rudimentary form of cellular "vision."

Mechanical perturbation of webbed edges in 3T3 cells.

We have previously described actin edge-bundles (AEBs) as cables of microfilaments lining the webbed edges of 3T3 cells (Zand and Albrecht-Buehler: Cell Motil. Cytoskeleton 13:195-211, 1989). We have suggested that AEBs, along with their cell-substratum adhesions, resist cortical tension and prevent the collapse of cytoplasm towards the nucleus. In this paper, we report several stages of AEB disassembly and re-formation induced by the following micro-manipulations: (1) Scoring of the webbed edge of a 3T3 cells with a micro-needle. As a result the sides of the score retracted and the severed AEB appeared to disassemble down to its terminal adhesion points. The retraction stopped after 20-40 seconds and the cells formed a webbed edge with large curvature. Over a period of 20-80 minutes, the new web decreased in length and depth, until it regained its approximate original shape. (2) Bending of cell processes at acute angles. As a result the processes moved until they projected at right angles to the side of the cell and formed new webs gradually expanded their area. In both cases, the nascent webs were lined by actin edge-bundles.

Surface extensions of 3T3 cells towards distant infrared light sources.

Using a specially designed phase-contrast light microscope with an infrared spot illuminator we found that approximately 25% of 3T3 cells were able to extend pseudopodia towards single microscopic infrared light sources nearby. If the cells were offered a pair of such light sources next to each other, 47% of the cells extended towards them. In the latter case 30% of the responding cells extended separate pseudopodia towards each individual light source of a pair. The strongest responses were observed if the infrared light sources emitted light of wavelengths in the range of 800-900 nm intermittently at rates of 30-60 pulses per min. The temperature increases of the irradiated spots can be shown to be negligible. The results suggest that the cells are able to sense specific infrared wavelengths and to determine the direction of individual sources.

Possible mechanisms of indirect gravity sensing by cells.

We have to distinguish between (a) direct gravisensing, in which specialized cells function as parts of a gravisensing organ and (b) indirect gravisensing, in which other cells that have no specialized gravity detectors are nevertheless affected by the inertial acceleration. In both cases, cells may detect (a) the direction of gravity ("up" versus "down"), and /or (b) the amplitude (0 - 1 g) of gravity. This chapter argues that the weight of single normal-sized cells (approximately 10 microns in diameter) is too small compared with other cellular forces to allow them the distinction between up and down. However, the weight of the surrounding medium is much larger. Cells may be able to sense certain environmental changes caused by gravity and thus may sense indirectly at least the amplitude of gravitational forces. In particular, the fluid environment of the cell can be expected at normal gravity to support microconvective currents that cease to flow at microgravity. Thus, the absence of gravity may be transduced into the accumulation of metabolites and ions from the cells and depletion of fresh nutrients. These changes, in turn, can affect the contacts of cells, their membrane potential, their cytoskeleton, and thus, ultimately, their behavior. As to ground-based simulations of microgravity, the above considerations suggest that the averaging of the vectorial force of gravity in clinorotation is inadequate for simulation because it may actually increase rather than suppress convective mixing above the normal levels.

The iris diaphragm model of centriole and basal body formation.

This paper suggests that the formation and structure of the microtubular skeleton of centrioles and basal bodies can be derived from the following simple geometric principle. A closed ring of nine microtubular initiation sites defines (1) a template for the packing of 18 additional microtubular initiation sites, and (2) the shape of nine rigid arms. Upon swivelling of each arm around a point located four initiation sites away on the initial ring, the array unfolds in a manner similar to the opening of an iris diaphragm. As a consequence, the curved shape of the microtubular triplet blades arises together with the clockwise rotational sense of the slanted blades of the centriole or basal body. The final diameter of the centriole (basal body) self-adjusts. Furthermore, the pitch of the triplet blades, the taper of centrioles and basal bodies, and the change of slant of the blades towards the distal end can be derived. In addition, the model points to a method of replication of pro-centrioles (pro-basal bodies). The hypothesis was tested by the fitting of electron microscopical cross sections of centrioles of 3T3 cells to the geometric shapes predicted by the model.

What structures, besides adhesions, prevent spread cells from rounding up?

The outline of cells in sparse cultures consists predominantly of concave and convex segments; straight segments are rare and ephemeral. The convex segments are areas of active cell expansion. The concave segments are stationary and web-shaped, similar in profile to the cables of a suspension bridge. In 3T3 fibroblasts, we have found a single microfilament bundle following the outline of every webbed edge and have called it the actin edge-bundle (AEB). While the AEB is composed predominantly of actin, alpha-actinin and myosin are also present. In contrast to normal stress fibers, AEBs are more resistant to several treatments that depolymerize F-actin. Once an AEB disassembles, however, the webbed edge collapses and retracts, suggesting that the actin edge-bundle is a specialized cytoskeletal structure that supports the webbed edges of interphase 3T3 fibroblasts. The stability of AEBs is independent of microtubules. We suggest that the microfilament bundles that frequently line the lateral contacts between epithelial cells in vivo may be related to the actin edge-bundle.

Long-term observation of cultured cells by interference-reflection microscopy: near-infrared illumination and Y-contrast image processing.

Interference-reflection microscopy (IRM) is the only method presently available with which to visualize cell-substratum adhesions in living tissue culture cells continuously for long periods of time without the use of fluorescent markers (Curtis: J. Cell Biol. 20:199-215, 1964; Izzard and Lochner: J. Cell Sci. 21:129-159, 1976). This method utilizes approximately 1% of the incident illumination to produce the IRM image (Verschueren: J. Cell Sci. 75:279-301, 1985) and so far has required the use of high-intensity light sources in the visible spectral range (400-800 nm). Unfortunately, visible light of this intensity and spectral range induces marked changes in the behavior and morphology of motile fibroblasts, including cessation of locomotion. In contrast, the present paper reports that continuous observations of live cells in IRM for periods of up to 8 hours are possible if the illuminating light is in the red to near-infrared range (650-950 nm) and without any observable change in normal cell morphology or behavior. In addition, we describe how the technique of Y-contrast image processing can be applied to IRM images to create a three-dimensional image of the ventral cell surface topography.

Rigidity of the nucleus during nuclear rotation in 3T3 cells.

Using near infrared microscopy and ultraviolet fluorescence microscopy of living 3T3 cells stained with the fluorochrome Hoechst 33342, we have demonstrated that the nucleoli and Hoechst 33342-stained chromocenters in the nucleus maintain a fixed pattern during nuclear rotation. We conclude that the term "nuclear rotation" refers to rotation of the entire nucleus in the cytoplasm of interphase cells, and that nuclear rotation is not an expression of karyoplasmic streaming. In conjunction with earlier results on nuclear rotation the data imply that the interface of nuclear rotation is located either between the two nuclear membranes or in the adjacent cytoplasm.

Distribution of multiple centrospheres determines migration of BHK syncitia.

After fusion of BHK cells with polyethylene glycol, the resulting syncitia contained in 77% of the cases multiple microtubule organizing centers (MTOCs), which were aggregated into a common centrosphere. Based on the observation of phagokinetic tracks, we found that the syncitia were able to locomote if the MTOCs aggregated into a common centrosphere cluster, and the clustered centrospheres were excluded from the cluster of nuclei of the syncitium. The results suggest that each individual pair of one nucleus and one centrosphere contributes, in a process of vectorial addition, its individual polarity to the polarity of the syncitium. Thus the widely accepted idea that the centrosphere is involved in the determination of cell polarity can be generalized beyond the case of single cells.

Role of cortical tension in fibroblast shape and movement.

In order to analyze the cellular mechanisms of shape formation, the shape of individual 3T3 cells was perturbed by micromanipulation resulting in the detachment and relaxation of a cellular extension and the bending of the extension to form an "elbow" at a variable angle beta. Finally, the tip of the extension was allowed to reattach to the substrate away from the cell. The cells reacted by drawing the extension tight. If beta less than 90 degrees, the elbow moved laterally for 8-15 min until the extension projected orthogonally at the cell surface. If beta greater than or equal to 90 degrees, the extension remained stationary. Finally, in all cases webs formed between attachment points in the perturbed area. If the tip of the extension was allowed to touch its own cell body, thus forming a loop, the cells invariably closed the loop. The paper interprets the cellular reaction as the result of cortical tension and suggests that it is a major factor in the formation of fibroblast shape and the expressions of fibroblast motility.

The reorganization of microtubules and microfilaments in differentiating keratinocytes.

Using immunofluorescence techniques, we have examined the microtubules and microfilaments in colonies of terminally differentiating human keratinocytes in tissue culture. The undifferentiated keratinocytes contained numerous microtubules, which radiated from a centrosomal organization center (MTOC). Differentiating keratinocytes, which leave the basal layer and begin to synthesize involucrin, displayed an altered cytoskeleton. Thick mats and coils of microtubules formed throughout the cytoplasm of the differentiated squames, and microfilaments were no longer visible after staining with phalloidin. Instead, only scattered stipples of phalloidin-stained material were observed. The results suggest that the terminal differentiation of epidermal cells involves a reorganization not only of the keratin filaments but of the entire cytoskeleton.

Light conductance in the ocular lens.

The collimated beam of a He-Ne laser aimed tangentially at the equator of submerged bovine or rabbit ocular lenses enters the lens and travels in the surface of the lens. We suggest that the effect is the result of light conductance along the interface between the bulk of the lens (including the lens epithelium on the anterior surface) as the medium of higher refractive index and the capsule as a medium of lower refractive index. Light conductance can be demonstrated in isolated lenses as well as in intact eyes. It is very sensitive to alterations of the state of the lens and therefore it may offer a new method to study very early stages of lens damage and cataract formation.

The degree of coupling of nuclear rotation in binucleate 3T3 cells.

In order to test the existence of mechanical coupling between the rotational movements of two adjacent nuclei, we prepared binucleate 3T3 cells and observed their nuclear movements by near infrared microscopy and recorded them with time-lapse video techniques. We found that 49 out of 110 (44%) of the selected binucleate cells expressed nuclear rotation. Rotation could occur in just one of the nuclei while the second nucleus remained stationary (31/110) or in both nuclei simultaneously (18/110). In almost all cases where both nuclei rotated simultaneously (15/110) they did so at different speeds and in opposite directions. The nuclei were observed to rotate in the same direction in only three of the examples. The results are consistent with a weak mechanical interaction between a rotating nucleus and its neighbor. Consistent with our previous observations in mononucleate cells, we did not find a characteristic position of the centrosphere or a special distribution of the microtubules or the intermediate filaments in binucleate cells with rotating nuclei. There was an absence of long, well-formed microfilament bundles beneath the nuclei during rotation, even in the local region beneath the rotating nucleus in those cells with one rotating and one stationary nucleus. Also consistent with observations of mononucleate cells, nuclear rotation was inhibited by treatment with colcemid, although the ability of the nuclei to rotate was eventually restored when the colcemid-containing medium was replaced with normal medium.