Dynamic assembly of surface structures in living cells

Xenopus kidney epithelial A6 cells form a monolayer with well established tight junctions (Fig. 2A) and numerous microvillar structures (Fig. 1). These microvilli are supported by the bundles of actin filaments extending from the cortical cytoskeleton (Fig. 1B). Time-lapse SICM imaging showed that these microvilli are dynamic structures (Fig. 2B). Even after 10 h of continuous observation, we have not noted any signs of slowing down of the microvillar dynamics. However, the addition of 1% formaldehyde to the bath medium effectively stopped any changes of microvillar shape (data not shown).

We observed that the microvillar density usually varied between different A6 cells within the same monolayer. By choosing a cell where the density of microvilli was low, allowing identification of individual microvilli, it was possible to quantify the dynamics of single microvilli (Fig. 2B). At any given time, some microvilli grew (marked orange), whereas the neighboring ones retracted (marked green). However, there was also a general tendency of the cell to retract (Fig. 2B Upper) or form (Fig. 2B Lower) microvilli at different periods of time. In the illustrated cell, the microvillar dynamics changed from retraction to formation during the 200 s separating the two sets of frames shown in Fig. 2B Upper and Lower. Often microvilli are formed along an axis (Fig. 2 A and B). Even those microvilli that did not grow or retract were not strictly “stable,” showing some lateral rearrangement (Fig. 2).

Initially, microvilli grew at a rate of about 5 nm/s (Fig. 2C). As the microvillus increased in height, the rate of growth decreased, which eventually resulted in the cessation of their growth (Fig. 2C). In contrast, the retraction of microvilli occurred at a rate of about 1.2 nm/s, which remained constant and independent on the microvilli height (Fig. 2E). The observed life cycle of a microvillus is depicted in Fig. 2D. After a short period of rapid growth, which characterizes a sudden appearance of a microvillus, a steady state ensues, followed by a retraction at a constant rate. An average duration of this cycle was found to be 12.1 ± 5.6 min (n = 244).

Formation of multiple aggregates of microvilli often led to ridge-like structures. Almost 50% of the cells in the monolayer had ridges rather than individual microvilli (Fig. 3A). It could be observed that the assembly of a ridge was initiated by the formation of a new microvillus alongside an existing one. Then the neighboring microvilli contact each other, forming relatively stable aggregates (Fig. 3E). Newly formed microvilli never associate together as a clump. They could associate only laterally to form a ridge. Sometimes these ridges angled at precisely 120°. The ridges had a unique height (within the boundaries of the normal distribution), whereas individual microvilli had a population of shorter microvilli (Fig. 3 B and D), which may reflect their formation or retraction (Fig. 3C).

To validate that the observed microvillus dynamics is not a specific property of a cultured cell line but rather a wide-ranging cellular phenomenon, we studied the microvillar dynamics in different cell types (including human cells). One example is epithelial nonsensory cells of the mouse organ of Corti. The well developed short microvilli of these cells could be considered predecessors of the stereocilia of sensory hair cells (5). In organotypic culture conditions (22), these epithelial supporting cells still possess a microvillar coating, albeit not as dense as in vivo (23). One type, Hensen's cells, is shown in Fig. 4A. Similar to A6 cells, time-lapse SICM observations revealed ongoing dynamics of microvilli on the surface of these Hensen's cells (Fig. 4B). Remarkably, the rates of microvilli formation and retraction in these mammalian epithelial cells were similar to those of the Xenopus A6 cells. In the example shown in Fig. 4B, it took about 3 min for the microvillus to form, which is very close to the values measured for A6 cells (Fig. 2C). Similar to A6 cells, the retraction of microvilli was slower. Analogous microvillar dynamics was also observed in another type of epithelial supporting cells of the organ of Corti, outer phalangeal cells (data not shown). As expected, the larger microvillar structures, stereocilia of sensory hair cells, were substantially more stable, showing no signs of dynamic changes of height or lateral arrangement within the accuracy of SICM imaging technique (data not shown). In addition, we performed time-lapse imaging of the surface of the following cell types: human colon cancer cells (CACO-2 line), murine melanocytes (melan-b line), breast cancer cells (T47D cell line), and fibroblasts (COS-7 cell line) (data not shown). In all these cells, we observed microvillar dynamics similar to those described above. We think, therefore, that these microvillar dynamics are likely to be a common phenomenon for short nonspecialized microvilli irrespective of their cell origin.

Several features of this dynamics should be noted. The initial formation rate of individual microvilli is about 5 nm/s, which is very close to the rate of elongation of the fertilization cone in sea urchin egg (5.5 nm/s) (24) and three to six times less than typical rates of filopodium extension (15–30 nm/s) (25). This is unlikely to be a simple coincidence. In contrast to filopodia, parallel actin fibers in microvilli and the fertilization cone are densely packed into bundles by different cross-linking proteins (26). The measured initial formation rate of these structures probably reflects the speed of assembly of the whole bundle. The rate of growth in our experiments shows a distinct inverse relationship with the height of the microvillus (Fig. 2C). This would be expected if formation of the microvillus depends on simple diffusion of actin and actin-binding proteins to its tip. As the microvillus grows, the longer diffusion path leads to a decrease in the rate of actin bundle assembly. A microvillus stops growing when the rate of formation becomes equal to the rate of ongoing actin depolymerization. Such dynamic steady-state equilibrium between actin bundle formation and actin depolymerization was proposed to regulate the length of large microvillar structures like Drosophila bristles (7), brush border microvilli (11), or stereocilia of hair cells (10). In our experiments, microvilli after formation were in a steady state for ≈5 min, followed by retraction with a constant rate of 1.2 nm/s (Fig. 2E), which is much slower than formation rate and is comparable to the rate of actin depolymerization at 2 nm/s (27). These data suggest that, at least in the retraction phase, actin polymerization is blocked and not involved in the control of the microvillar height. However, it is not clear what signal controls retraction or growth of a microvillus. Presumably this on/off signal has to operate locally, because two neighboring microvilli may exist in different phases of their life cycle (Fig. 3C).

It has been proposed that microvilli serve as “the archetypal factory of an actin bundle” (26), constantly providing a cell with organized bundle assemblies, which could be used by the cell to form larger specialized microvillar structures. Here we have shown that microvilli are constantly changing, which indeed implies ongoing assembly and disassembly or utilization of actin bundles. In fact, the dynamics of actin assembly in cortical cytoskeleton (28) is strikingly similar to the microvillar dynamics observed in our experiments. Moreover, microvillar dynamics also provide clusters of microvilli, which serve as crystallization points for more stable surface structures, where the microvillar steady state is prolonged. Therefore, we conclude that microvilli may serve as elementary “building blocks” of the larger structures on the surface of the cell.

This paper was submitted directly (Track II) to the PNAS office.

This work was supported by the Biotechnology and Biological Science Research Council, the Office of Naval Research, and the Wellcome Trust.