Cell Biology: Social Distancing of Microtubule Ends Increases Their Assembly Rates

Abstract

Microtubules assembled from artificial centrosomes in microfluidic chambers of defined size are amenable to high resolution live imaging of their dynamics and space-filling properties. By using this experimental regime in conjunction with cytoplasmic extract, a new study finds that microtubule end density negatively influences their assembly rates.

The female ovum is generally considered to be the largest cell, by volume, in the body of many metazoans (excepting the largest motor neurons in large animals, and syncytial tissues). On average, ova range from ~50 microns to over 3 millimeters in diameter in some amphibians. Human ova settle in at about 100 microns in diameter, making them hundreds of times larger by volume than a majority of the other cells in the body. This presents an interesting problem for microtubules because in large cells they may be compelled to assemble into accordingly longer polymers in order to fill the cytoplasm of the cell and fulfill transport, nuclear positioning, spindle positioning and other microtubule-dependent activities. These extra-long microtubules would have to assemble, presumably, by utilizing kinetic parameters and tubulin dimer concentrations similar to those that would be found in much smaller cells. How this occurs is not fully understood, but a number of key reviews have nicely summarized the problem [1,2].

In this issue of Current Biology, Geisterfer and colleagues [3] have cleverly adapted hydrogel lithography [4] to produce chambers of defined size and volume into which they can perfuse Xenopus egg cytoplasmic extract [5]. The chambers used in Geisterfer et al. [3] span sizes from 30 to 115 microns in diameter, enabling the authors to investigate microtubule dynamics in a closed system that is physiologically relevant in size and volume to metazoan cells. The authors use this technology to study factors that contribute to cytoskeletal size scaling in closed ‘cells’ of defined volumes.

Using fluorescent EB1 to label growing microtubule ends, and Aurora A-coated beads as artificial microtubule organizing centers (aMTOCs), the authors observed an interesting mildly positive correlation between microtubule assembly rates and chamber volume. Further parsing of the data showed a much stronger negative correlation between microtubule assembly rates and EB1 density (Figure 1A). This parameter can be scored within a chamber by evaluating the number of EB1 comets within a circle of defined radius around each EB1 comet (Figure 1B). The positive correlation between microtubule assembly and chamber size might be explained by a lower overall density of EB1-decorated microtubule ends in chambers of larger size. A simple but perhaps naive mechanism has been suggested by previous studies that have shown a negative correlation between the rate of microtubule nucleation at the centrosome and microtubule assembly rates. In general, greater numbers of nucleated microtubules were correlated with lower rates of microtubule assembly [6,7]. However, Geisterfer and colleagues measured nucleation as a function of EB1 comets that cross a defined radius outward from the aMTOC and found no correlation between overall nucleation rate and the chamber size except in the very smallest of chambers (30 microns in diameter) [3].

Figure 1. Diagram of microfluidic flow chamber filled with cytosolic extract.

(A) Regions with denser spacing of microtubule ends assembled more slowly than regions with sparse ends. Speculation that microtubule assembly factors are depleted from regions with dense microtubule ends (purple region). (B) Quantification of microtubule end density. (C) Hypothetical depletion of factors (purple) with dual nucleation and assembly promoting activities during branched microtubule assembly [17]. MT, microtubule.

If the average nucleation rate is invariant between chambers, what then explains the variations in assembly rate between chambers and also within a chamber? Here is where the improved imaging capabilities of this technology significantly enhance previous size scaling studies accomplished by enclosing cytosol in oil droplets of defined sizes [8]. Using higher resolution imaging afforded by the microperfusion chambers, the authors reveal that within regions of high EB1 density, microtubule assembly is slowed. Both size scaling (how microtubules fill large cytoplasmic volumes) and subcellular spatial variation in microtubule assembly (how microtubules assemble differently from others within the same cytoplasm) are important mysteries in cytoskeletal regulation. Both of these problems can be interrogated at a physiological scale using the microperfusion chambers.

In the present paper, the authors theorize that a putative microtubule assembly factor might be locally depleted in regions of high microtubule end density, leading to reduced rates of assembly. Local depletion, of GTP-tubulin would surely slow assembly; however, existing studies are converging on the idea that the diffusion of tubulin dimers in cytosol might be too rapid to experience local depletion, especially in the larger chambers [9,10]. Instead, the authors favor local depletion of an assembly factor, such as XMAP215, to explain their observations. Its proposed elongated shape [11,12] may indeed impede its diffusion throughout the cytosol, leading to local depletion and supporting a diffusion-limited mechanism to explain the negative relationship between microtubule density and growth rates. Perhaps even more significantly, XMAP215 represents an interesting choice because its bourgeoning role as a microtubule nucleator might facilitate its titration away from microtubule ends [13–16]. Titration has the potential to become an even more significant contributor if microtubules in these microfluidic chambers elongate using branched nucleation [17,18]. This hypothesis is illustrated in Figure 1C. Future studies will determine if this is a viable mechanistic hypothesis.

In conclusion, the technology for rapidly enclosing proteins and cytosolic extracts into defined chambers amenable to imaging will significantly enhance the identification of factors that influence the kinetics of cytoskeletal assembly and disassembly. Such chambers can be used to interrogate the impact of cell geometry on cytoskeletal dynamics and other cellular activities. They can model the ‘closed system’ of a vertebrate cell while eliminating the heterogeneity and cell shape disruptions common to adherent mammalian culture systems. Finally, these microfluidic chambers, which can be prepared in a variety of shapes and sizes, provide an excellent means to investigate the constraints that cellular geometry and volume place on cytoplasmic processes.