Intramolecular strain coordinates kinesin stepping behavior along microtubules.
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ABSTRACT:
SUBMITTER: Yildiz A
PROVIDER: S-EPMC2613771 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Two structurally distinct filamentous tracks, namely singlet microtubules in the cytoplasm and axonemes in the cilium, serve as railroads for long-range transport processes in vivo In all organisms studied so far, the kinesin-2 family is essential for long-range transport on axonemes. Intriguingly, in higher eukaryotes, kinesin-2 has been adapted to work on microtubules in the cytoplasm as well. Here, we show that heterodimeric kinesin-2 motors distinguish between axonemes and microtubules. Unlike canonical kinesin-1, kinesin-2 takes directional, off-axis steps on microtubules, but it resumes a straight path when walking on the axonemes. The inherent ability of kinesin-2 to side-track on the microtubule lattice restricts the motor to one side of the doublet microtubule in axonemes. The mechanistic features revealed here provide a molecular explanation for the previously observed partitioning of oppositely moving intraflagellar transport trains to the A- and B-tubules of the same doublet microtubule. Our results offer first mechanistic insights into why nature may have co-evolved the heterodimeric kinesin-2 with the ciliary machinery to work on the specialized axonemal surface for two-way traffic.
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Project description:Trafficking of the proteins that form gap junctions (connexins) from the site of synthesis to the junctional domain appears to require cytoskeletal delivery mechanisms. Although many cell types exhibit specific delivery of connexins to polarized cell sites, such as connexin32 (Cx32) gap junctions specifically localized to basolateral membrane domains of hepatocytes, the precise roles of actin- and tubulin-based systems remain unclear. We have observed fluorescently tagged Cx32 trafficking linearly at speeds averaging 0.25 ?m/s in a polarized hepatocyte cell line (WIF-B9), which is abolished by 50 ?M of the microtubule-disrupting agent nocodazole. To explore the involvement of cytoskeletal components in the delivery of connexins, we have used a preparation of isolated Cx32-containing vesicles from rat hepatocytes and assayed their ATP-driven motility along stabilized rhodamine-labeled microtubules in vitro. These assays revealed the presence of Cx32 and kinesin motor proteins in the same vesicles. The addition of 50 ?M ATP stimulated vesicle motility along linear microtubule tracks with velocities of 0.4-0.5 ?m/s, which was inhibited with 1 mM of the kinesin inhibitor AMP-PNP (adenylyl-imidodiphosphate) and by anti-kinesin antibody but only minimally affected by 5 ?M vanadate, a dynein inhibitor, or by anti-dynein antibody. These studies provide evidence that Cx32 can be transported intracellularly along microtubules and presumably to junctional domains in cells and highlight an important role of kinesin motor proteins in microtubule-dependent motility of Cx32.
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Project description:Mammalian KIF3AC is classified as a heterotrimeric kinesin-2 that is best known for organelle transport in neurons, yet in vitro studies to characterize its single molecule behavior are lacking. The results presented show that a KIF3AC motor that includes the native helix α7 sequence for coiled-coil formation is highly processive with run lengths of ∼1.23 μm and matching those exhibited by conventional kinesin-1. This result was unexpected because KIF3AC exhibits the canonical kinesin-2 neck-linker sequence that has been reported to be responsible for shorter run lengths observed for another heterotrimeric kinesin-2, KIF3AB. However, KIF3AB with its native neck linker and helix α7 is also highly processive with run lengths of ∼1.62 μm and exceeding those of KIF3AC and kinesin-1. Loop L11, a component of the microtubule-motor interface and implicated in activating ADP release upon microtubule collision, is significantly extended in KIF3C as compared with other kinesins. A KIF3AC encoding a truncation in KIF3C loop L11 (KIF3ACΔL11) exhibited longer run lengths at ∼1.55 μm than wild-type KIF3AC and were more similar to KIF3AB run lengths, suggesting that L11 also contributes to tuning motor processivity. The steady-state ATPase results show that shortening L11 does not alter kcat, consistent with the observation that single molecule velocities are not affected by this truncation. However, shortening loop L11 of KIF3C significantly increases the microtubule affinity of KIF3ACΔL11, revealing another structural and mechanistic property that can modulate processivity. The results presented provide new, to our knowledge, insights to understand structure-function relationships governing processivity and a better understanding of the potential of KIF3AC for long-distance transport in neurons.
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Project description:Synaptic plasticity often involves changes in the structure and composition of dendritic spines. Vesicular cargos and organelles enter spines either by exocytosing in the dendrite shaft and diffusing into spines or through a kinesin to myosin hand-off at the base of spines. Here we present evidence for microtubule (MT)-based targeting of a specific motor/cargo pair directly into hippocampal dendritic spines. During transient MT polymerization into spines, the kinesin KIF1A and an associated cargo, synaptotagmin-IV (syt-IV), are trafficked in unison along MTs into spines. This trafficking into selected spines is activity-dependent and results in exocytosis of syt-IV-containing vesicles in the spine head. Surprisingly, knockdown of KIF1A causes frequent fusion of syt-IV-containing vesicles throughout the dendritic shaft and diffusion into spines. Taken together, these findings suggest a mechanism for targeting dendritic cargo directly into spines during synaptic plasticity and indicate that MT-bound kinesins prevent unregulated fusion by sequestering vesicular cargo to MTs.
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Project description:Kinesin-1 is an ATP-driven, processive motor that transports cargo along microtubules in a tightly regulated stepping cycle. Efficient gating mechanisms ensure that the sequence of kinetic events proceeds in the proper order, generating a large number of successive reaction cycles. To study gating, we created two mutant constructs with extended neck-linkers and measured their properties using single-molecule optical trapping and ensemble fluorescence techniques. Owing to a reduction in the inter-head tension, the constructs access an otherwise rarely populated conformational state in which both motor heads remain bound to the microtubule. ATP-dependent, processive backstepping and futile hydrolysis were observed under moderate hindering loads. On the basis of measurements, we formulated a comprehensive model for kinesin motion that incorporates reaction pathways for both forward and backward stepping. In addition to inter-head tension, we found that neck-linker orientation is also responsible for ensuring gating in kinesin.
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Project description:Heterodimeric KIF3AC and KIF3AB, two members of the mammalian kinesin-2 family, generate force for microtubule plus end-directed cargo transport. However, the advantage of heterodimeric kinesins over homodimeric ones is not well-understood. We showed previously that microtubule association for entry into a processive run was similar in rate for KIF3AC and KIF3AB at ?7.0 ?m-1 s-1 Yet, for engineered homodimers of KIF3AA and KIF3BB, this constant is significantly faster at 11 ?m-1 s-1 and much slower for KIF3CC at 2.1 ?m-1 s-1 These results led us to hypothesize that heterodimerization of KIF3A with KIF3C and KIF3A with KIF3B altered the intrinsic catalytic properties of each motor domain. Here, we tested this hypothesis by using presteady-state stopped-flow kinetics and mathematical modeling. Surprisingly, the modeling revealed that the catalytic properties of KIF3A and KIF3B/C were altered upon microtubule binding, yet each motor domain retained its relative intrinsic kinetics for ADP release and subsequent ATP binding and the nucleotide-promoted transitions thereafter. These results are consistent with the interpretation that for KIF3AB, each motor head is catalytically similar and therefore each step is approximately equivalent. In contrast, for KIF3AC the results predict that the processive steps will alternate between a fast step for KIF3A followed by a slow step for KIF3C resulting in asymmetric stepping during a processive run. This study reveals the impact of heterodimerization of the motor polypeptides for microtubule association to start the processive run and the fundamental differences in the motile properties of KIF3C compared with KIF3A and KIF3B.
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Project description:Kinesins are well known to power diverse long-range transport processes in virtually all eukaryotic cells. The ATP-dependent processive stepping as well as the regulation of kinesin' activity have, thus, been the focus of extensive studies over the past decades. It is widely accepted that kinesin motors can self-regulate their activity by suppressing the catalytic activity of the "heads." The distal random coil at the C terminus, termed "tail domain," is proposed to mediate this autoinhibition; however, a direct regulatory influence of the tail on the processive stepping of kinesin proved difficult to capture. Here, we simultaneously tracked the two distinct head domains in the kinesin-2 motor using dual-color super resolution microscopy (dcFIONA) and reveal for the first time their individual properties during processive stepping. We show that the autoinhibitory wild-type conformation selectively impacts one head in the heterodimer but not the other. Our results provide insights into the regulated kinesin stepping that had escaped experimental scrutiny so far.
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