Overexpression of (β)-actin is known to alter cell morphology, though its effect on cell motility has not been documented previously. Here we show that overexpressing (β)-actin in myoblasts has striking effects on motility, increasing cell speed to almost double that of control cells. This occurs by increasing the areas of protrusion and retraction and is accompanied by raised levels of (β)-actin in the newly protruded regions. These regions of the cell margin, however, show decreased levels of polymerised actin, indicating that protrusion can outpace the rate of actin polymerisation in these cells. Moreover, the expression of (β)*-actin (a G244D mutant, which shows defective polymerisation in vitro) is equally effective at increasing speed and protrusion. Concomitant changes in actin binding proteins show no evidence of a consistent mechanism for increasing the rate of actin polymerisation in these actin overexpressing cells. The increase in motility is confined to poorly spread cells in both cases and the excess motility can be abolished by blocking myosin function with butanedione monoxime (BDM). Our observations on normal myoblasts are consistent with the view that they protrude by the assembly and cross linking of actin filaments. In contrast, the additional motility shown by cells overexpressing (β)-actin appears not to result from an increase in the rate of actin polymerisation but to depend on myosin function. This suggests that the additional protrusion arises from a different mechanism. We discuss the possibility that it is related to retraction-induced protrusion in fibroblasts. In this phenomenon, a wave of increased protrusion follows a sudden collapse in cell spreading. This view could explain why it is only the additional motility that depends on spreading, and has implications for understanding the differences in locomotion that distinguish tissue cells from highly invasive cell types such as leucocytes and malignant cells.
We have previously shown that addition of type 1 transforming growth factor-beta (TGFbeta1) to an exponentially growing population of mink lung CCl64 cells increases their average intermitotic time from 14.4 to 20.3 hours, predominantly by extending G1 from 7.5 to 13.5 hours. Here we have used the DRIMAPS system (digitally recorded interference microscopy with automatic phase-shifting) for obtaining data on cellular mass distribution, cell motility and morphology. We found no significant change in the cells' rate of mass increase following TGFbeta1 treatment, which implies that the treated cells attained a higher mass during their extended cell cycle and this was confirmed by direct measurement of cell size. However, the cells showed a dramatic motile response to treatment: TGFbeta1-treated cells had a significantly higher time-averaged speed of 36.2 microm hour-1 compared to 14.5 microm hour-1 for the control cells. The time course of the response was gradual, reaching a maximum mean speed of 52.6 microm hour-1 after 15 hours exposure. We found that the gradual onset of the response was probably not due to a slow accumulation of a secondary factor but because cells were dividing throughout the experiment and most of the response to TGFbeta1 occurred only after the first cell division in its presence. Thus, taking only those cells that had not yet divided, the time-averaged speed of treated cells (26.1 micrometer hour-1) was only moderately higher than that of untreated cells (14.9 micrometer hour-1) whereas, for those cells that had divided, the difference in speed between treated cells (45.1 micrometer hour-1) and untreated cells (14.1 microm hour-1) was much greater. Increased speed was a consequence of enhanced protrusion and retraction of the cell margin coupled with an increase in cell polarity. TGFbeta1 also increased the mean spreading of the cells, measured as area-to-mass ratio, from 3.2 to 4.4 micrometer2 pg-1, and the intracellular mass distribution became more asymmetric. The observations indicate that a G2 signal may be necessary to reach maximal motility in the presence of TGFbeta1.
Using data automatically acquired by microinterferometry from large numbers of chick fibroblasts, we have detected rapid microfluctuations in the rates of protrusion and retraction of the cell margin which were strongly suppressed by colcemid, nocodazole and taxol. Fluctuations in the rate of retraction of the margin were about twice as powerful as fluctuations in the rate of protrusion. High-frequency fluctuations were also apparent in the cell track and in measures of cell spreading, shape and speed. These rapid fluctuations were also all suppressed by colcemid and nocodazole, sometimes by doses insufficient to disrupt the majority of microtubules. Taxol on the other hand did not suppress fluctuations in direction of the cell track nor fluctuations in the spreading of the cells but it was very effective at suppressing variations in protrusion and retraction and in cell speed and shape. We discovered that much slower, larger-scale variations in protrusion, retraction, spreading, shape and speed resulted from the accumulation of these rapid, microtubule-dependent fluctuations of the cell margin. These large-scale variations in cell behaviour were also suppressed by the same drug treatments that were effective in suppressing the corresponding high-frequency fluctuations. We speculate that a function of microtubules is to enhance the fibroblast's responses to its environment by causing microfluctuations of the cell's margin which give rise to large-scale variations in cell behaviour.
A new technique of microinterferometry permits cellular growth and motile dynamics to be studied simultaneously in living cells. In isolated chick heart fibroblasts, we have found that the non-aqueous mass of each cell tends to increase steadily, with minor fluctuations, throughout the cell cycle. The spread area of each cell also tends to increase during interphase but fluctuates between wide limits. These limits are dependent on the cell's mass and the upper limit is particularly sharp and directly proportional to mass. From a dynamical point of view, the spread area of a cell is determined by the balance between the rates of two antagonistic processes: protrusion of cellular material into new territory and retraction of material from previously occupied territory. The spatial asymmetry of these processes determines the translocation of the cell. We have found with the chick fibroblasts that the rates of the two processes are generally closely matched to each other and appear to be dependent on the cell's area of spreading. Both continue incessantly in well spread cells, even when there is no net translocation of the cell, and the lower limit of each activity is directly proportional to spread area. The two processes show different behaviour, however, during changes in the spread area of the cell. Both increases and decreases in area appear to be brought about by changes in the rate of retraction, the rate of protrusion remaining relatively constant. A simple stochastic model based on a limited supply of adhesion molecules can simulate all our observations including the mass-limited spreading, the strong correlation between protrusion and retraction and the retraction-dominated changes in area. We conclude that the spread area of the cell is actively regulated, possibly by a simple automatic mechanism that adjusts the area of spreading in relation to the mass of the cell and controls the rate of protrusion to compensate rapidly for spontaneous fluctuations in retraction.
A new form of chamber for studying chemotaxis, similar in principle to the Zigmond chamber, allows the behaviour of the cells in a linear concentration gradient to be observed directly. The chamber was developed mainly for studying chemotaxis in fibroblasts using interferometric microscopy and the main design criteria were that it should have better optical characteristics, a higher dimensional precision and better long-term stability than the Zigmond chamber. It is made entirely from glass by grinding a blind circular well centrally in the counting platform of a Helber bacteria counting chamber. This procedure leaves an annular ‘bridge’, approximately 1 mm wide, between the new inner circular well and the original outer annular well. This bridge fulfils the same function as the linear bridge of the Zigmond chamber but the precise construction of the counting chamber ensures that a gap of 20 microns between bridge and coverslip can be accurately and repeatedly achieved when the chamber is assembled. It is envisaged that the improved optical clarity, dimensional accuracy and long-term stability of the new chamber will be advantageous in other applications, particularly in studies requiring critical microscopy or a precise knowledge of the gradient and in studies of cells, such as fibroblasts, that move much more slowly than neutrophils.