Kinesin-II is required for axonal transport of choline acetyltransferase in Drosophila - PubMed (original) (raw)
Kinesin-II is required for axonal transport of choline acetyltransferase in Drosophila
K Ray et al. J Cell Biol. 1999.
Abstract
KLP64D and KLP68D are members of the kinesin-II family of proteins in Drosophila. Immunostaining for KLP68D and ribonucleic acid in situ hybridization for KLP64D demonstrated their preferential expression in cholinergic neurons. KLP68D was also found to accumulate in cholinergic neurons in axonal obstructions caused by the loss of kinesin light chain. Mutations in the KLP64D gene cause uncoordinated sluggish movement and death, and reduce transport of choline acetyltransferase from cell bodies to the synapse. The inviability of KLP64D mutations can be rescued by expression of mammalian KIF3A. Together, these data suggest that kinesin-II is required for the axonal transport of a soluble enzyme, choline acetyltransferase, in a specific subset of neurons in Drosophila. Furthermore, the data lead to the conclusion that the cargo transport requirements of different classes of neurons may lead to upregulation of specific pathways of axonal transport.
Figures
Figure 1
The protein sequence of KLP64D is homologous to KIF3A of mouse and KRP85 of sea urchin. The identical sequences are shown as white letters in black background, whereas functionally similar ones are marked with gray boxes in the background. Multiple sequence alignments were done using the pileup program of the GCG analysis package with default options set. Sequence identities were highlighted using Box shade 3.21 shareware provided by the ISREC Bioinformatics Group (http://www-isrec.unil.ch/isrec.htm). The location of Klp64D mutant changes are noted.
Figure 2
KLP64D gene expression pattern revealed by RNA in situ hybridization with a specific probe encoding the COOH terminus made from a 1.6 kb Spe I/HindIII fragment, labeled with digoxygenin-dUTP, and visualized using alkaline phosphatase activity linked to an antidigoxygenin antiserum. Bars: A, 100 μm (A, B, and D); C, 25 μm. (A) Arrows indicate staining in the developing CNS of a stage 12/13 embryo when germ band retraction is partly complete. The anterior side of the embryo is towards the left and the ventral side is down. (B) The expression in the PNS is first detected at late stage 17 of embryogenesis, after the completion of germ band retraction and dorsal closure. Most of the neuronal development is complete by this time. The small arrows indicate staining in the lateral chordotonal (lch) neurons. All eight lch5 cells of the abdominal segments and one lch3 in the third thoracic segment are visible. The arrowheads indicate dorsal chordotonal organs in each of the abdominal segments. The large arrow indicates the optic lobe in the CNS. (C) An enlarged view of lateral pentascolopedial chordotonal organs (lch5) of a late stage 17 embryo stained by the KLP64D probe revealed the cytoplasmic outline of the cells. There are five lch5 neurons clustered together at the basal side of the epidermis. (D) Staining in the brain of a third instar larva. A low level of expression is seen in the cells of the ventral ganglion (arrowheads) and in the optic lobe (arrow). Arrows indicate the staining in the optic lobe region where inputs from the bolwig nerve as well as the photoreceptor neurons of the larval eye disc is received. A faint staining is also observed in the crescent shaped developing lamina region of the optic lobe.
Figure 3
Immunostaining pattern of rabbit anti-KLP68D (A, C, and E) and anti-HRP (B, D, and F) in the epidermis of a late stage 16 embryo. The anterior side of the embryo in all the figures is placed towards the left and the dorsal side is upwards. The KLP68D antiserum stained neuronal soma of a subset of sensilla while anti-HRP marked all the sensory neurons in the epidermis (Jan and Jan 1982). Sense organ nomenclature is followed as in Campos-Ortega and Hartenstein 1997. Bars: A, 50 μm (A–D); E, 10 μm (E and F). (A) The anterior part of the embryo is shown, including the first two thoracic segments. Relatively strong staining of the dorsal (dch3) and lateral (lch3) chordotonal organ neurons in the first and second thoracic segments is evident. The polyinnervated external sense organ (vbd) and external papilla (Py) in the second and first thoracic segments, respectively, are only weakly stained. Similarly, the dorsal (do) or antennalles organ and the terminal organ (to) or maxillarorgan are weakly stained. Most of the sensory cells of these two organs are just beyond the plane of focus. The faint staining observed in a group of cells (indicated by an asterisk) in the dorsal region of the first thoracic segment are identified as bolwig organ. (B) The same region as in A labeled with goat anti-HRP, which stains all neuronal cell bodies and processes and clearly shows that only a subset of neurons are stained for KLP68D. (C) Staining in the abdominal segments is shown. The cell bodies of the lateral (lch5) and the ventral (vch1x2) chordotonal neurons are relatively strongly stained. The neuronal cell bodies of ventral papillae (vp4 and vp5) and the lateral trichoid sensillum (lh2) are less intensely stained. In addition, there is weak staining in some unrecognized cells in between vp4 and vp5. (D) Same field as in C stained with anti-HRP. (E) A high magnification image of a region of the lateral epidermis containing the pentascholopedial organ (lch5) and other sensilla. Staining is restricted to the neuronal cytoplasm of the lch5. (F) The same field as in E stained with anti-HRP, which highlights all the neurons. Identified neurons are marked in the figure. The neurons of the monoinnervated external sensilla, lh1 and lp2, and the multi-dendritic neurons, ltd and lda, are not stained significantly relative to lch5.
Figure 4
Segmental nerve bundles from a Klc1/Df(3L)8ex94 third instar larva, simultaneously stained with either mouse anti-ChAT (A) and rabbit anti-KLP68D (B) or with anti-ChAT (C and E) and rabbit anti-SYT (D and F). They are visualized using FITC anti–mouse and Cy5 anti–rabbit. Both frames are simultaneously excited and captured by separate photomultipliers in a single optical scan of 1-μm thickness. Accumulations of the respective antigens in the axons in individual clogs are marked by different sized arrows and arrowheads. Both KLP68D and ChAT antisera usually stain the same foci (clogs) in the nerve roots (see Table for details). SYT generally does not associate (arrowheads in C and D) with the ChAT in these foci although occasional coincident staining is seen (arrows in E and F). Bar, 10 μm.
Figure 5
Coimmunoprecipitation analysis. Immunoprecipitation from wild-type (WT) and mouse KIF3A expressing (3A+) Drosophila using K2.4 mAb raised against sea urchin KRP85, affinity-purified anti-KLP68D, or nonspecific Ig. Western blots of immunoprecipitates were probed with K2.4 or affinity-purified anti-KLP68D.
Figure 6
Staining of various markers of axonal transport in the cell bodies and axons of heterozygous (A and C) and homozygous (B and D) klp64D mutant larval brains and segmental nerves. Samples are stained with mouse anti-ChAT (A–D), XTRITC-coupled α-bungarotoxin (E and F) and with mouse anti-CSP antibody (G and H), respectively. A few were also stained with rabbit antiactin antibody (C′ and D′). Bars: C′, 10 μm (C, C′, D, and D′); F, 100 μm (A, B, E–H). (A) _Klp64Dk1/_TM3 third instar larva ventral ganglion has anti-ChAT staining confined to the neuropil. The punctate nature of the staining indicates localization of ChAT in synaptic bulbs. There was no staining in the cell cortex (arrowhead) or in the nerve roots (arrows). (B) The homozygous _Klp64Dk1_third instar larval brain exhibited significant ChAT staining in the axons (arrows) of the nerve roots and in the cell cortex (arrowheads). Abnormally strong staining can also be seen at the boundary of the neuropil. (C and C′) A part of a segmental nerve bundle is shown from a Klp64Dk1/TM3 larva stained with mouse anti-ChAT and rabbit antiactin antibody, respectively. Both cases showed no specific accumulation of these two antigens in any of the axons. (D and D′) The equivalent part of a similar nerve bundle from a Klp64Dk1 homozygous larva showed strong accumulations of ChAT antigen in some of the axons (arrows). The antiactin staining in the axons is slightly higher in intensity than the control, but it is not selectively higher in the axons that showed accumulated ChAT as shown in F. E–H show equivalent regions of ventral ganglia from Klp64Dk1/TM3 (E and G), and the Klp64Dk1 homozygous third instar larvae, respectively. They are stained with α-bungarotoxin (E and F) and anti-CSP (G and H) and the pattern is similar between the control and mutants.
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