The kinesin-8 Kip3 depolymerizes microtubules with a collective force-dependent mechanism

Abstract

Microtubules are highly dynamic filaments with dramatic structural rearrangements and length changes during the cell cycle. An accurate control of the microtubule length is essential for many cellular processes in particular, during cell division. Motor proteins from the kinesin-8 family depolymerize microtubules by interacting with their ends in a collective and length-dependent manner. However, it is still unclear how kinesin-8 depolymerizes microtubules. Here, we tracked the microtubule end-binding activity of yeast kinesin-8, Kip3, under varying loads and nucleotide conditions using high-precision optical tweezers. We found that single Kip3 motors spent up to 200 s at the microtubule end and were not stationary there but took several 8-nm forward and backward steps that were suppressed by loads. Interestingly, increased loads, similar to increased motor concentrations, also exponentially decreased the motors' residence time at the microtubule end. On the microtubule lattice, Kip3 had a different binding behavior suggesting that the observations are distinct for the microtubule end. The force dependence of the end residence time enabled us to estimate what force must act on a single motor to achieve the microtubule depolymerization speed of a motor ensemble. This force is higher than the stall force of a single Kip3 motor, supporting a collective force-dependent depolymerization mechanism. Understanding the mechanics of kinesin-8's microtubule end activity will provide important insights into cell division with implications for cancer research. STATEMENT OF SIGNIFICANCEKinesin-8 motors are important for microtubule length regulation and overexpressed in different types of cancer. Yet, on the molecular level, it is unclear how these motors depolymerize microtubules. Using high-precision optical tweezers, we measured how single yeast kinesin-8 motors, Kip3, interacted with the microtubule end. Interestingly, we found that single Kip3 motors were still motile at the microtubule end. The force dependence of how long single motors were associated with the microtubule end enabled us to estimate what force motors must exert onto each other to achieve the collective microtubule depolymerization speed of many motors. Our data support a collective force-dependent depolymerization mechanism. A better understanding of Kip3's microtubule end activity has implications for cell division and associated diseases.

Single depolymerizing and transport kinesins stabilize microtubule ends

“…For kinesin-8, another contribution may stem from the strongly bound state at the microtubule end (Arellano-Santoyo et al, 2017) associated with only slow forward and backward stepping (Bugiel et al, 2020).…”

“…Reflected light waves interfere and create a dark microtubule image on a brighter background (see Simmert et al, 2018 for details). Inset: Exemplary IRM image of a microtubule (scale bar: 2 μm) allowed us to performed microtubule length measurements over a long period (60 min) without inducing photodamage (Bugiel et al, 2020). We measured the microtubule depolymerization speed as the total length change-the difference between the microtubule length in the first and last recorded image-divided by the total acquisition time.…”

“…Consistent with the simulation, depolymerization by kinesin-8 occurred at higher motor numbers at the end that result in traffic jams on individual protofilaments (Figure S2). These traffic jams are required for the collective, force-dependent depolymerization mechanism (Bugiel et al, 2020) that unifies the so-called "bump-off" (Varga et al, 2009) and "switching" (Arellano-Santoyo et al, 2017) models.…”

“…e.g., Du, English, & Ohi, 2010;Edzuka & Goshima, 2019;Fukuda, Luchniak, Murphy, & Gupta, 2014;Glunči c et al, 2015;Gupta, Carvalho, Roof, & Pellman, 2006;Klemm, Bosilj, Glunči c, Pavin, & Toli c, 2018;Mayr et al, 2007;Niwa et al, 2012;Rizk, DiScipio, Proudfoot, & Gupta, 2014;Stumpff, von Dassow, Wagenbach, Asbury, & Wordeman, 2008;Su, Ohi, & Pellman, 2012;Tischer, Brunner, & Dogterom, 2009;Varga et al, 2006;Varga, Leduc, Bormuth, Diez, & Howard, 2009). The budding yeast kinesin-8 Kip3 is a slow and weak, plus-enddirected kinesin that depolymerizes microtubules in a collective, lengthand force-dependent manner (Bugiel, Chugh, Jachowski, Schäffer, & Jannasch, 2020;Gupta et al, 2006;Jannasch, Bormuth, Storch, Howard, & Schäffer, 2013;Varga et al, 2006Varga et al, , 2009. For depolymerization, it is essential that Kip3 reaches the microtubule end.…”

“…The high run length originates from a weakly bound slip state (Jannasch et al, 2013;Sudhakar et al, 2021) in combination with a second microtubule binding site located in the tail domain (Mayr, Storch, Howard, & Mayer, 2011;Su et al, 2011). This binding site also enhances Kip3's extraordinary long residence time at the microtubule end and enables Kip3 to crosslink and slide antiparallel microtubules apart (Bugiel et al, 2020;Dave et al, 2018;Roostalu & Surrey, 2013;Su et al, 2013;Wargacki, Tay, Muller, Asbury, & Davis, 2010).…”