Kinesin-1 tail autoregulation and microtubule-binding regions function in saltatory transport but not ooplasmic streaming

Abstract

The N-terminal head domain of kinesin heavy chain (Khc) is well known for generating force for transport along microtubules in cytoplasmic organization processes during metazoan development, but the functions of the C-terminal tail are not clear. To address this, we studied the effects of tail mutations on mitochondria transport, determinant mRNA localization and cytoplasmic streaming in Drosophila. Our results show that two biochemically defined elements of the tail - the ATP-independent microtubule-binding sequence and the IAK autoinhibitory motif - are essential for development and viability. Both elements have positive functions in the axonal transport of mitochondria and determinant mRNA localization in oocytes, processes that are accomplished by biased saltatory movement of individual cargoes. Surprisingly, there were no indications that the IAK autoinhibitory motif acts as a general downregulator of Kinesin-1 in those processes. Time-lapse imaging indicated that neither tail region is needed for fast cytoplasmic streaming in oocytes, which is a non-saltatory bulk transport process driven solely by Kinesin-1. Thus, the Khc tail is not constitutively required for Kinesin-1 activation, force transduction or linkage to cargo. It might instead be crucial for more subtle elements of motor control and coordination in the stop-and-go movements of biased saltatory transport.

Fig. 1.

Khc tail mutations inhibit Kinesin-1-driven axonal transport. (A) Locations of missense mutations in the Khc tail. The sequence of the C-terminal region of Drosophila Khc (codons 780-975) is shown with changes caused by the tail alleles (Khc⁷⁶, Khc²², Khc⁷⁷, 4R-A) noted above. Coil 3 of the stalk (III, dashed line) includes binding sites for Klc, Milton and their associated cargoes (Klc). Coil 4a-b (IV, dashed line) includes a cargo binding site in Neurospora (N.c. Cargo). Coil 4c (dotted line) includes part of a tail microtubule-binding region (Microtubule) that extends towards a conserved IAK motif (“IAK”) that can interact with the N-terminal Khc head to inhibit force generation. (B) Fluorescence images of nerves in third instar larvae stained with anti-CSP. Images are representative of 10-15 larvae per genotype, either wild type (+) or the indicated Khc allele over the Khc²⁷ null allele. (C) z-projections of axon terminals on muscles 6/7 in segment A3 of control (+) and Khc⁷⁷/Khc²⁷ (77) mutant larvae. Larvae expressing mitochondria-targeted GFP (mitoGFP) in neurons (green in merge) were stained for Khc (red in merge) and Fasciclin (Fas). Scale bars: 30 μm in B; 20 μm in C.

Fig. 2.

Khc IAK motif mutations inhibit axonal transport of mitochondria. Drosophila larvae of the indicated Khc genotypes expressing mitoGFP in neurons were anesthetized and segmental nerves imaged by time-lapse confocal microscopy (see Movies 1-3 in the supplementary material). (A) The number of mitochondria per minute that moved anterograde or retrograde (flux) into a photobleached zone in one segmental nerve per larva was counted for ten animals per genotype (mean ± s.e.m.). All mutant means were significantly lower than that of wild type (P<0.05). (B) The percentage of time that anterograde-moving mitochondria spent in anterograde runs relative to total tracking time, which included pauses and reverse runs, is on the left. Mean anterograde run lengths are on the right. Values were determined by digital tracking in five larvae per genotype (mean ± s.e.m.). Sample size (number of mitochondria tracked) is noted in each bar.

Fig. 3.

Khc IAK motif mutations inhibit body axis patterning in oocytes. (A) Fluorescent in situ hybridization images for osk mRNA in wild-type (left) and Khc⁷⁷ mutant (right) Drosophila oocytes at the indicated stages of oogenesis. The arrow indicates a lagging central concentration of osk in stage-9 mutants. Scale bars: 25 μm in stage 8; 50 μm in stages 9 and 10A. (B) Eggs from female germline clones homozygous for the Khc alleles indicated on the left were scored for defects in dorsal appendage morphology that indicate defective dorsal-ventral patterning.

Fig. 4.

IAK and tail microtubule-binding mutations do not inhibit Khc-dependent ooplasmic streaming. (A) Fluorescent yolk endosomes were imaged by time-lapse microscopy in stage-10B Drosophila oocytes homozygous for the indicated Khc alleles. Panels show time projections of 15 frames each (225 seconds total). Moving endosomes appear in the projections as streaks or trails of spots. The arrow indicates a residual circular area of streaming in a Khc¹⁷ head mutant oocyte (compare Movies 4 and 5 in the supplementary material). By contrast, streaming appeared normal in tail mutants (compare Movies 4 and 6 in the supplementary material). (B) Time projections of oocytes homozygous for a Khc null allele without any Khc transgene (27), with a wild-type Khc transgene (4R), or with a mutant Khc transgene that has four arginine codons converted to alanines in the tail microtubule-binding region (4R-A). Streaming patterns were normal in the 4R-A mutant. (C) Time projections of slow streaming in stage-9 oocytes homozygous for the indicated tail alleles. Motion appeared normal in the mutants. Scale bars: 40 μm. (D) Mean velocities (+ s.e.m.) of moving yolk endosomes in stage-10B and stage-9 oocytes homozygous for the Khc alleles indicated on the x-axis. Ten randomly selected yolk endosomes were tracked in each of five oocytes per genotype. Sample sizes were 45-50 moving endosomes. Asterisks indicate values significantly different from the wild type as determined by general linear modeling (P<0.05).