Kinesin-14 motors participate in a force balance at microtubule plus-ends to regulate dynamic instability

Abstract

Kinesin-14 molecular motors represent an essential class of proteins that bind microtubules and walk toward their minus-ends. Previous studies have described important roles for Kinesin-14 motors at microtubule minus-ends, but their role in regulating plus-end dynamics remains controversial. Kinesin-14 motors have been shown to bind the EB family of microtubule plus-end binding proteins, suggesting that these minus-end–directed motors could interact with growing microtubule plus-ends. In this work, we explored the role of minus-end–directed Kinesin-14 motor forces in controlling plus-end microtubule dynamics. In cells, a Kinesin-14 mutant with reduced affinity to EB proteins led to increased microtubule lengths. Cell-free biophysical microscopy assays were performed using Kinesin-14 motors and an EB family marker of growing microtubule plus-ends, Mal3, which revealed that when Kinesin-14 motors bound to Mal3 at growing microtubule plus-ends, the motors subsequently walked toward the minus-end, and Mal3 was pulled away from the growing microtubule tip. Strikingly, these interactions resulted in an approximately twofold decrease in the expected postinteraction microtubule lifetime. Furthermore, generic minus-end–directed tension forces, generated by tethering growing plus-ends to the coverslip using λ-DNA, led to an approximately sevenfold decrease in the expected postinteraction microtubule growth length. In contrast, the inhibition of Kinesin-14 minus-end–directed motility led to extended tip interactions and to an increase in the expected postinteraction microtubule lifetime, indicating that plus-ends were stabilized by nonmotile Kinesin-14 motors. Together, we find that Kinesin-14 motors participate in a force balance at microtubule plus-ends to regulate microtubule lengths in cells.

Force-transducing molecular ensembles at growing microtubule tips control mitotic spindle size

“…To complete the system, we added microtubule tip tracking protein EB3, which is known to bind HSET in a way that makes microtubule binding domains on both EB3 and HSET available for engaging microtubules. 47,48 We verified this interaction and confirmed that it allowed HSET to transport EB away from microtubule tips on individual microtubules (Figure S2A/Video S2). Unexpectedly, however, when both minus-end directed motor and tip tracker were present in our assay, we observed not only pulling, but also pushing forces as was indicated by the increase in the distance between the beads (Figure 1C,D and Figure S1).…”

“…We showed that a simple system consisting of a single minus-end directed motor that acts synergetically with growing dynamic microtubules via the action of tip tracking EB proteins can perform both, separation of the two microtubule asters and stabilization of their bipolar organization. HSET is known to affect the aster formation, the spindle size and to interact with EB proteins 15,17,18,48 , and EBs are known to make multivalent condensates and facilitate transport 38,39,45,46 . Here, we reveal how the combined EB/HSET/microtubule system allows harnessing microtubule forces that stabilize the bipolar organization of two asters, which is not achievable by mechanisms used by these molecules alone.…”

Microtubule acetylation dyshomeostasis in Parkinson’s disease

“…Tau helps in MT bundling and promotes its growth and inhibits its shrinkage by regulating the labile domain of MT [ 20 ]. A recent study by Mecak et al [ 21 ] proved the involvement of kinesin-14 motor proteins in regulating the dynamic instability of MTs. The MT cytoskeleton is constantly exposed to external mechanical forces arising from active ongoing processes within or outside the cell.…”

Cik1 and Vik1 accessory proteins confer distinct functions to the kinesin-14 Kar3

“…The activity of Kar3 heterodimers on MTs was first described as a minus-end-directed translocation with low processivity ( Endow et al, 1994 ), a property common to other kinesin-14 family members. Subsequent Kar3 studies provided evidence for its potential to switch between plus-end- and minus-end-directed movement ( Molodtsov et al, 2016 ), or to stably associate with a growing plus-end ( Sproul et al, 2005 ), possibly when in complex with Bim1, the budding yeast homolog of MAPRE family members (also known as EB1) ( Kornakov et al, 2020 ; Ogren et al, 2022 ; Sproul et al, 2005 ). In addition, although early work with Kar3 provided evidence for plus-end depolymerase activity ( Sproul et al, 2005 ), later work failed to document this activity ( Mieck et al, 2015 ).…”