Reconstitution of Kinesin-1 Activation

Abstract

Autoinhibition is an important regulatory mechanism for cytoskeletal motor proteins. Kinesin-1 (kinesin hereafter), the ubiquitous plus-end directed microtubule motor, is thought to be controlled by a complicated autoinihibition mechanism, but the molecular details remain unclear. Conformational changes mediated by intramolecular interactions between the C-terminal tail and N-terminal motor domains of the kinesin heavy chain (KHC) are proposed to be one facet of motor regulation. The dimeric KHC also binds two copies of the kinesin light chains (KLCs), which have been implicated in both autoinhibition and cargo-dependent activation of the tetrameric motor complex, although the precise mechanisms remain opaque. Using in vitro reconstitution, we show that the KLC strongly inhibits the kinesin-microtubule interaction via an independent mechanism from the tail-motor interaction within KHC. Kinesin cargo-adaptor proteins that bind KLC activated processive movement of the kinesin tetramer but the landing rate of these activated complexes remained low. The addition of MAP7, which specifically binds to the KHC, strongly enhanced activated motor motility by dramatically increasing the landing rate and processivity of the activated kinesin motors. Our results support a model whereby the activity of the kinesin tetramer is regulated by independent tail- and KLC-based inhibition mechanisms, and that cargo-adaptor binding to the KLC directly releases both of these inhibitions. However, we find that a third component, a non-motor MAP is required for robust activity of the activated motor. Thus, human kinesin activity is regulated by a two-factor mechanism comprised of intramolecular allosteric regulation, as well as intermolecular kinesin-adaptor and kinesin-MAP interactions.

An ALS-associated KIF5A mutant forms oligomers and aggregates and induces neuronal toxicity

“…To study the molecular mechanism of ALS caused by KIF5A mutations, we expressed mScarlet fused KIF5A (mSca::KIF5A) and mScarlet fused KIF5A(Δexon27) (mSca::KIF5A(Δexon27)) in a neuron-like cell line CAD (Qi, Wang, McMillian, & Chikaraishi, 1997) (Fig 1B-E). mSca::KIF5A was mostly diffuse in the cell but about 30 % mSca::KIF5A-expressed cells showed small aggregates in the cytoplasm (Fig 1C and E) in agreement with our prior finding showing a propensity of KIF5A to form oligomers (Chiba, Ori-McKenney, Niwa, & McKenney, 2022). The ratio of cells showing aggregation is higher compared with cells expressing mScarlet fused KIF5B, a homologue of KIF5A (Hirokawa et al, 2009).…”

“…To examine the effect of Δexon27 mutation on the interaction with KLC1 and oligomerization, we next purified heavy chain dimers (KIF5A) and Kinesin-1 tetramers (KIF5A-KLC1) in the presence or absence of Δexon27 mutation. Firstly, we expressed full-length KIF5A::mSca and KIF5A(Δexon27)::mSca in sf9 cells and purified them by affinity chromatography and size exclusion chromatography (SEC) (Chiba et al, 2022). In the SEC, wild type KIF5A predominantly eluted in a single peak (Fig 3A, blue shaded area).…”

KLC4 shapes axon arbors during development and mediates adult behavior

“…Kinesin-1 can bind MTs via the KHC C-terminal tail and crosslink or bundle MTs (Navone et al, 1992;Seeger and Rice, 2010). Binding of KLC to KHC releases the autoinhibition of KHC and can tip the balance between kinesin-1's cargo transport activities and the ability of the KHC tail region to bind MTs (Cai et al, 2007;Chiba et al, 2021;Wong and Rice, 2010). Our data are consistent with a model in which KLC4 promotes the increased MT dynamics or severing that allows MT branch invasion.…”

Pathogenic mutations in the chromokinesin KIF22 disrupt anaphase chromosome segregation

“…Rather than physically interacting with the motor domain, it is also possible that structural changes in the tail of KIF22 could have allosteric effects on the motor domain. An allosteric mechanism by which conformational changes are propagated down the stalk to the motor domain has recently been proposed to contribute to the inactivation of kinesin-1 motors by kinesin light chain, which binds the tail (Chiba, Ori-McKenney, Niwa, & McKenney, 2021). KIF22 inactivation may be caused by altered motor domain mechanochemistry, which changes in the tail could affect allosterically and modification of a2 could affect directly.…”