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. 1998 Apr 20;141(2):443-54.
doi: 10.1083/jcb.141.2.443.

Kinesin light chains are essential for axonal transport in Drosophila

J G Gindhart Jr  1 , C J Desai, S Beushausen, K Zinn, L S Goldstein
Affiliations

Kinesin light chains are essential for axonal transport in Drosophila

J G Gindhart Jr et al. J Cell Biol. .

Abstract

Kinesin is a heterotetramer composed of two 115-kD heavy chains and two 58-kD light chains. The microtubule motor activity of kinesin is performed by the heavy chains, but the functions of the light chains are poorly understood. Mutations were generated in the Drosophila gene Kinesin light chain (Klc), and the phenotypic consequences of loss of Klc function were analyzed at the behavioral and cellular levels. Loss of Klc function results in progressive lethargy, crawling defects, and paralysis followed by death at the end of the second larval instar. Klc mutant axons contain large aggregates of membranous organelles in segmental nerve axons. These aggregates, or organelle jams (Hurd, D.D., and W.M. Saxton. 1996. Genetics. 144: 1075-1085), contain synaptic vesicle precursors as well as organelles that may be transported by kinesin, kinesin-like protein 68D, and cytoplasmic dynein, thus providing evidence that the loss of Klc function blocks multiple pathways of axonal transport. The similarity of the Klc and Khc (. Cell 64:1093-1102; Hurd, D.D., and W.M. Saxton. 1996. Genetics 144: 1075-1085) mutant phenotypes indicates that KLC is essential for kinesin function, perhaps by tethering KHC to intracellular cargos or by activating the kinesin motor.

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Figures

Figure 1
Figure 1
(A) Map of Klc genomic interval and location of Klc mutants used in this analysis. The Klc transcription unit is composed of six exons, and is transcribed from right to left in this diagram. Ptp69D (Desai et al., 1996) is located 3′ of Klc, and is transcribed toward Klc. Map coordinates are shown in kb. Restriction sites are shown as follows: P, PstI; R, EcoRI; N, the NotI site in cosmid 8.1 used for subcloning GEN-KLC (see Materials and Methods). The location of P[lacW] in Klc intron 1 is shown as an upward pointing arrow. Sequences deleted by the lesions Df(3L)8ex94 and Df(3L)8ex34 are shown as solid lines. The endpoints of Df(3L)8ex94 are shown; the endpoints of Df(3L)8ex34 are unknown. (B) Maps of KLC transgenic constructs MYC-KLC and GEN-KLC. The MYC-KLC transgene is composed of KLC amino acids 1–508 fused to a 13–amino acid epitope tag recognized by the anti-MYC monoclonal antibody 9E10 (Evan et al., 1985). This fusion protein is under the transcriptional control of the Drosophila polyubiquitin promoter, which ensures high levels of transgene expression in all tissues (Lee et al., 1988). The map position of the NotI-EcoRI genomic DNA fragment included in GEN-KLC is also shown.
Figure 2
Figure 2
(A–C). Klc mutant larvae have locomotion defects that are rescued by GEN-KLC. Anterior is to the left, and the dorsal surface of the larva is shown. (A) Wild-type and Df(3L)8ex94/+ (shown) larvae crawl using peristaltic waves of muscle contraction. Most if not all of the ventral surface remains on the medium. (B) Klc mutant larvae of the genotype Df(3L)8ex94/Klc 1 (shown) and other hypomorphic Klc mutant combinations flip their posterior end off the surface during crawling. This phenotype is quite similar to the tail flipping phenotype observed in certain Khc mutant combinations (Hurd and Saxton, 1996). (C) GEN-KLC rescues the tail-flipping phenotype. GEN-KLC; Df(3L)8ex94/Klc 1 larvae exhibit normal larval crawling behavior (compare wild-type [A] to [C] rescued larvae). The MYC-KLC transgene also rescues the Klc-dependent tail flipping phenotype (not shown). (D) Characterization of KLC antisera at different Drosophila life stages. Total protein was extracted from wild-type individuals at the following life stages: 0–6 h embryos (lane 1); third instar larvae (lane 2); adult females (lane 3); adult males (lane 4); and adult males transformed with MYC-KLC (lanes 5 and 6). Lanes 1–5 were incubated with affinity-purified KLC antisera, and lane 6 was incubated with an antibody recognizing the epitope tag of MYC-KLC. The epitope tag causes MYC-KLC to have an apparent molecular weight slightly larger than 58 kD, the molecular weight of Drosophila KLC (Gauger and Goldstein, 1993). Lane 6 shows that the epitope-specific antibody recognizes a protein of the same apparent molecular weight as KLC (lane 5) in MYC–KLC transformants. The adult-specific 91-kD protein may not be a KLC isoform, as it is recognized by antisera specific to KLC-LG3. peptide coupled to KLH, but not other KLC antisera (see Materials and Methods). Molecular weight standards are shown in kD (right).
Figure 3
Figure 3
Loss of Klc function causes disruption of axonal transport. Larvae heterozygous for Klc (A, B) or Klc 1/Df(3L)8ex94 mutants (C, D) were simultaneously incubated with antibodies specific for cysteine string protein (A, C) and synaptotagmin (B, D) to compare the accumulation patterns of these synaptic vesicle components in wild-type and Klc mutant backgrounds. Although accumulation of CSP and SYT is punctate but fairly uniform in the segmental nerves of control larvae (A, B), large aggregates of CSP and SYT immunoreactivity are observed in Klc mutants (C, D), suggesting that normal axonal transport of these proteins are blocked. Furthermore, the pattern of CSP (C) accumulation completely overlaps SYT (D), suggesting that these proteins are found in the same subset of organelle jams. Individual organelle jams are presumed to be confined to a single axon (Hurd and Saxton, 1996). Bar, 25 μM.
Figure 4
Figure 4
KLP68D and cytoplasmic dynein are found in organelle jams. Segmental nerves of Df(3L)8ex94/Klc 1 larvae were incubated with antibodies to CSP (A) and KLP68D (B), or SYT (C) and DHC, the motor component of cytoplasmic dynein (D). Both KLP68D and DHC immunoreactivity are observed in organelle jams. Bar, 20 μM.
Figure 5
Figure 5
KHC and KLC accumulate in Klc-dependent organelle jams. Df(3L)8ex94/ Klc 1 larvae were incubated with antibodies to CSP (A, C) and antibodies to KHC (B) and KLC (D). KHC aggregates are observed in the segmental nerves of Klc mutant larvae. This observation would suggest that KHC can bind and transport intracellular cargoes in a KLC-independent manner; however, KLC protein accumulation is also observed in Klc mutant segmental nerves (D). Bar, 20 μM.
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