The growth speed of microtubules with XMAP215-coated beads coupled to their ends is increased by tensile force

Abstract

The generation of pulling and pushing forces is one of the important functions of microtubules, which are dynamic and polarized structures. The ends of dynamic microtubules are able to form relatively stable links to cellular structures, so that when a microtubule grows it can exert a pushing force and when it shrinks it can exert a pulling force. Microtubule growth and shrinkage are tightly regulated by microtubule-associated proteins (MAPs) that bind to microtubule ends. Given their localization, MAPs may be exposed to compressive and tensile forces. The effect of such forces on MAP function, however, is poorly understood. Here we show that beads coated with the microtubule polymerizing protein XMAP215, the Xenopus homolog of Dis1 and chTOG, are able to link stably to the plus ends of microtubules, even when the ends are growing or shrinking; at growing ends, the beads increase the polymerization rate. Using optical tweezers, we found that tensile force further increased the microtubule polymerization rate. These results show that physical forces can regulate the activity of MAPs. Furthermore, our results show that XMAP215 can be used as a handle to sense and mechanically manipulate the dynamics of the microtubule tip.microtubule polymerase | microtubule dynamics | optical trap X MAP215 is a microtubule plus-end binding protein that alters microtubule dynamics (1-4) by promoting microtubule growth. It was identified as a factor increasing microtubule polymerization rates in Xenopus egg extracts (5). In vivo studies have shown that disruption of XMAP215 function leads to short interphase microtubules, reduced microtubule growth rate, and increased frequency of microtubule catastrophe and pause events (6-11). Depletion of XMAP215 also results in short, abnormally formed mitotic spindles and short astral microtubules (7,12,13). In vitro studies have demonstrated that XMAP215 binds preferentially to the microtubule plus end and follows the growth and shrinkage of the microtubule tip, catalyzing microtubule growth (14-16). In addition to localization at the microtubule plus end and centrosomes (17-19), proteins of the XMAP215/Dis1 family are found at the kinetochores (9, 20, 21), which are sites of high force during cell division. Microtubules themselves can exert pulling and pushing forces (22)(23)(24)(25)(26)(27)(28)(29)(30). Therefore, the localization of XMAP215 at microtubule ends suggests that XMAP215 can function under load. However, the effect of forces on XMAP215 activity has not been directly analyzed. In this study, we investigated the influence of forces-directed toward the microtubule plus and minus end-on XMAP215 activity in vitro. Microtubule growth and shrinkage were monitored with high spatial and temporal resolution by visualizing the position of XMAP215-coated polystyrene microspheres bound to the tips of dynamic microtubules. The microspheres, so-called "beads," served as handles to apply forces on XMAP215 molecules using optical tweezers. Results XMAP215-Coated Beads Remain Attached to Growing ...

Light-emitting self-assembled peptide nucleic acids exhibit both stacking interactions and Watson–Crick base pairing

“…Because the crystal unit cell has a volume of 11,676 Å 3 (Supplementary Table 1) and contains eight molecules, we can estimate that 4.11 × 10 8 di-PNA building-block molecules organize into the ordered structures each second. In comparison to other rapid elongating systems of a natural origin, such as the microtubule that elongates by an average rate of 0.66 µm per minute 28 , the assembly kinetics of the di-PNA is over 20 times faster. When the same experiment was carried out in an open environment, instead of a sealed capillary, the elongation rate was about ten times faster due to evaporation of the solution, leading to higher local concentrations of PNA.…”

The Kinesin-8 Kip3 Switches Protofilaments in a Sideward Random Walk Asymmetrically Biased by Force

“…Carboxylated polystyrene microspheres (mean diameter 0.59 mm, Bangs Lab, Fishers, IN) were coated covalently with a 3 kDa polyethylene glycol linker and a green fluorescent protein (GFP) antibody as described previously (28,31). The GFP antibody was expressed and purified in the protein expression facility of the Max Planck Institute for Molecular Cell Biology and Genetics (Dresden, Germany).…”

Versatile microsphere attachment of GFP-labeled motors and other tagged proteins with preserved functionality

“…Subsequently, we tested for specific binding using different GFP-labeled motor proteins: truncated rat kinesin-1 (his 6 -rkin430-eGFP), budding yeast kinesin-8 (his 6 -Kip3-eGFP, his 6 -eGFP-Kip3, Kip3-his 6 -eGFP), human kinesin-8 (his 6 -Kif18A-eGFP), and orphan (ungrouped) kinesin Kip2 (his 6 -Kip2-GFP, his 6 -GFP-Kip2). Furthermore, we coupled the microspheres to the microtubule-polymerizing protein XMAP215-GFP-his 7 as described in [30]. For all proteins, we observed functional activity via microtubule interactions.…”

“…Furthermore in one experiment, we shock-froze the microspheres with bound XMAP215 (no single-molecule conditions) and kept them at -80°C. After 6 months, the microspheres still showed functionality [30]. The column "Motility" describes the fraction of motile microspheres.…”