Single depolymerizing and transport kinesins stabilize microtubule ends

Abstract

Microtubules are highly dynamic cellular filaments and an accurate control of their length is important for many intracellular processes like cell division. Among other factors, microtubule length is actively modulated by motors from the kinesin superfamily. For example, yeast kinesin-8, Kip3, motors depolymerize microtubules by a cooperative, force-and length-dependent mechanism. However, whether single motors can also depolymerize microtubules is unclear. Here, we measured how single kinesin motors influenced the stability of microtubules in an in vitro assay. Using label-free interference reflection microscopy, we determined the spontaneous microtubule depolymerization rate of stabilized microtubules in the presence of kinesins. Surprisingly, we found that both single Kip3 and nondepolymerizing kinesin-1 transport motors, used as a control, stabilized microtubules further. For Kip3, this behavior is contrary to the collective force-dependent depolymerization activity of multiple motors. Because of the control measurement, the finding may hint at a more general stabilization mechanism. The complex, concentration-dependent interaction with microtubule ends provides new insights into the molecular mechanism of kinesin-8 and its regulatory function of microtubule length.

A Quick and Reproducible Silanization Method by Using Plasma Activation for Hydrophobicity‐Based Kinesin Single Molecule Fluorescence–Microscopy Assays

“…To determine the motility parameters of single kinesin motors, we generated kymographs showing the position of the kinesin along the microtubule axis as a function of time (Figure 5 A). Based on the kymographs and fits to the histograms (Figure 5 B), the speed and the run length was μm/s and μm (mean±fit error, ), consistent with our own[ 14 , 50 , 51 ] and literature values. [ 52 , 53 ] Thus, the air+PP plasma activated surfaces did not change the performanceof kinesins and was optimally suited for stepping assays.…”

“…To determine the motility parameters of single kinesin motors, we generated kymographs showing the position of the kinesin along the microtubule axis as a function of time (Figure 5A). Based on the kymographs and fits to the histograms (Figure 5B), the speed and the run length was 0:8 � 0:1 μm/s and 0:76 � 0:04 μm (mean � fit error, N ¼ 49), consistent with our own [14,50,51] and literature…”

Approaches to visualize microtubule dynamics in vitro

“…Другие научные группы тоже начинают использовать IRM микроскопию по протоколу из работы [39]. В статье [44] для изучения способности отдельных молекул кинезина вызывать деполимеризацию микротрубочек был сделан выбор в пользу IRM из-за необходимости проведения длительных записей процесса медленной деполимеризации микротрубочек, стабилизированных с помощью GMPCPP. В работе [45] с помощью IRM изучались эффекты C-концевого хвоста α-мономера тубулина на динамику микротрубочки, а в работе [46]…”