Pavarotti/MKLP1 Regulates Microtubule Sliding and Neurite Outgrowth in Drosophila Neurons

Abstract

Summary Recently, we demonstrated that kinesin-1 can slide microtubules against each other providing the mechanical force required for initial neurite extension in Drosophila neurons. This sliding is only observed in young neurons actively forming neurites and is dramatically downregulated in older neurons. The downregulation is not caused by the global shut-down of kinesin-1, as the ability of kinesin-1 to transport membrane organelles is not diminished in mature neurons, suggesting that microtubule sliding is regulated by a dedicated mechanism [1]. Here, we have identified the “mitotic” kinesin Pavarotti (Pav-KLP) as an inhibitor of kinesin-1-driven microtubule sliding. Depletion of Pav-KLP in neurons strongly stimulated the sliding of long microtubules and neurite outgrowth, while its ectopic overexpression in the cytoplasm blocked both of these processes. Furthermore, postmitotic depletion of Pav-KLP in Drosophila neurons in vivo reduced embryonic/larval viability, with only a few animals surviving to the third instar larval stage. A detailed examination of motor neurons in the surviving larvae revealed the overextension of axons and mistargeting of neuromuscular junctions, resulting in uncoordinated locomotion. Taken together, our results identify a new role for Pav-KLP as a negative regulator of kinesin-1 driven neurite formation. These data suggest an important parallel between long microtubule-microtubule sliding in anaphase B and sliding of interphase microtubules during neurite formation.

Role of kinesin-1–based microtubule sliding in Drosophila nervous system development

“…Previously, we have reported kinesin-1-based MT sliding in tissue culture cells and culture neurons (6,7,9). Here, we demonstrate that robust MT sliding occurs in neurons in vivo.…”

“…We have shown that kinesin-1 is required for MT sliding in cultured neurons and kinesin-1 depletion inhibits both neurite outgrowth and regeneration (7,8). Additionally, we have observed MT sliding in axons as well as MTs pushing on the axon tip (9). Recent studies from other groups have also implicated MT translocation in axon extension and dendritic organization (10)(11)(12).…”

“…Either of these defects will impair neuronal communication, potentially resulting in similar phenotypic outputs: locomotion defects and eventual lethality. In support of this idea, depletion of the MT-sliding regulator, Pavarotti/MKLP1, results in locomotion and viability defects (9).…”

“…To test whether the C-terminal MT-binding site of KHC is necessary for MT sliding, we assayed MT sliding using α-tubulin tagged with a tandem dimer of the photoconvertible probe EOS (tdEOS-tubulin) (9,19), which changes emission spectra from green to red upon exposure to UV light (20). To quantify MT sliding, we expressed tdEOS-tubulin in Drosophila S2 cells, photoconverted a small subset of MTs from green to red, and imaged converted MTs in the red channel.…”

Microtubule–microtubule sliding by kinesin-1 is essential for normal cytoplasmic streaming in Drosophila oocytes

“…Cytoskeletal filaments and molecular motors are major players that generate the forces required for these activities. For instance, kinesin-1 and microtubules have been shown to provide the mechanical forces required in multiple cellular contexts, such as organelle transport (1)(2)(3)(4), ooplasmic streaming (5)(6)(7)(8)(9), and process formation (10)(11)(12)(13)(14)(15).…”