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Nature Neuroscience | Article

UNC-33 (CRMP) and ankyrin organize microtubules and localize kinesin to polarize axon-dendrite sorting

Journal name:
Nature Neuroscience
Volume:
15,
Pages:
48–56
Year published:
DOI:
doi:10.1038/nn.2970
Received
Accepted
Published online

Abstract

The polarized distribution of neuronal proteins to axons and dendrites relies on microtubule-binding proteins such as CRMP, directed motors such as the kinesin UNC-104 (Kif1A) and diffusion barriers such as ankyrin. The causative relationships among these molecules are unknown. We show here that Caenorhabditis elegans CRMP (UNC-33) acts early in neuronal development, together with ankyrin (UNC-44), to organize microtubule asymmetry and axon-dendrite sorting. In unc-33 and unc-44 mutants, axonal proteins were mislocalized to dendrites and vice versa, suggesting bidirectional failures of axon-dendrite identity. unc-44 directed UNC-33 localization to axons, where it was enriched in a region that resembled the axon initial segment. unc-33 and unc-44 were both required to establish the asymmetric dynamics of axonal and dendritic microtubules; in their absence, microtubules were disorganized, the axonal kinesin UNC-104 invaded dendrites, and inappropriate UNC-104 activity randomized axonal protein sorting. We suggest that UNC-44 and UNC-33 direct polarized sorting through their global effects on neuronal microtubule organization.

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  1. unc-33 mutants mislocalize presynaptic proteins to dendrites.
    Figure 1: unc-33 mutants mislocalize presynaptic proteins to dendrites.

    (a,b) PVD neuron morphology. (a) des-2::myristoyl::GFP marker in an L4 worm; cb, PVD cell body. (b) Diagrams of PVD morphology. The unbranched ventral process is the axon. (c,d) Representative images of RAB-3::mCherry and SAD-1::GFP in PVD neurons of wild-type (c) and unc-33(mn407) (d) worms, with schematic diagrams of phenotypes as in b. For each set of fluorescence micrographs, top panel is the maximum intensity projection of dendritic focal planes and bottom panel is the maximum intensity projection of axonal focal planes. White and yellow arrowheads, axonal and dendritic puncta, respectively; cb, PVD cell body; asterisks, gut autofluorescence. Anterior is at left and dorsal is up in all panels. Scale bars, 10 μm. (e,f) Quantification of fraction of worms with qualitative defects in axonal localization (e) and dendritic mislocalization (f) of RAB-3::mCherry and SAD-1::GFP (n > 30 worms per genotype). Error bars, standard error of proportion (s.e.p.). Alternative quantification of fluorescence intensity per worm is provided in Supplementary Figure 1. (g) unc-33 gene structure showing exons (black boxes), introns (lines), untranslated regions (gray boxes) and lesions in unc-33 alleles. Arrows denote the positions of start codons for alternative unc-33 transcripts. Three UNC-33 protein isoforms (long, medium and short) are depicted with predicted microtubule-assembling domains (MT) in blue and length in amino acids (aa). (h) Predicted microtubule-assembling domain of UNC-33, with rat CRMP-2. Gray regions, conserved residues; asterisk, the conserved glutamate mutated to lysine in unc-33(ky880).

  2. UNC-33L functions in PVD during the establishment of polarity.
    Figure 2: UNC-33L functions in PVD during the establishment of polarity.

    (a,b) Isoform-specific and cell-autonomous rescue of axonal protein localization in PVD neurons of unc-33 mutants. Quantification of axonal localization defects (a) and dendritic mislocalization defects (b) of RAB-3::mCherry and SAD-1::GFP were as in Figure 1; n > 25 worms per genotype. (c–e) Development of PVD neurons, visualized with des-2–driven myrisotyl::GFP marker, with schematic diagrams at right. In c, two focal planes are shown for a late L2/early L3 stage worm to show the two neurites being elaborated. White arrowheads, the primary processes of PVD; cb, PVD cell body; asterisks, gut autofluorescence. Anterior is at left and dorsal is up in all panels. Scale bars, 10 μm. (f) Quantification of axonal protein localization in PVD after heat shock–driven UNC-33L expression in unc-33(mn407) mutants (n > 25 worms per condition), with a corresponding developmental time line. Time is given in hours after release from L1 starvation. Rescue was defined as correct localization of RAB-3::mCherry in adult PVD neurons. All error bars indicate s.e.p.

  3. UNC-33L is enriched in PVD axons.
    Figure 3: UNC-33L is enriched in PVD axons.

    (a) UNC-33L immunoreactivity in nerve ring of wild-type worm. (b) Biologically active UNC-33L::GFP protein expressed from a pan-neuronal promoter in a wild-type worm, showing localization in nerve ring axons and absence from sensory dendrites. nr, nerve ring. (c–e) Representative images of UNC-33L::GFP, UNC-33S::GFP and UNC-33L N terminus (N-term)::GFP proteins in wild-type PVD neurons, with schematic diagrams at right. Red brackets, region of UNC-33L enrichment in axon; arrowheads, expression in ventral nerve cord; black brackets, proximal segment of PVD axon used for comparing fluorescence intensities in f. Scale bar in c also applies to d,e. (f) Quantification of UNC-33L::GFP, UNC-33S::GFP and GFP fluorescence, expressed as ratio of axon initial domain to axon proximal domain. Error bars, s.e.m. ***P < 0.001, Bonferroni t-test; NS, not significant. Anterior is at left and dorsal is up in all panels. Scale bars, 10 μm.

  4. unc-104 kinesin mislocalizes presynaptic proteins to dendrites in unc-33 mutants.
    Figure 4: unc-104 kinesin mislocalizes presynaptic proteins to dendrites in unc-33 mutants.

    (a–d) Representative images of RAB-3::mCherry and SAD-1::GFP in PVD neurons of wild-type (a), unc-104(e1265) (b), unc-33(ky880) (c) and unc-104; unc-33 (d) worms, with corresponding diagrams. For each set of fluorescence micrographs, top panel is the maximum intensity projection of dendritic focal planes and bottom panel is the maximum intensity projection of axonal focal planes. White and yellow arrowheads, axonal and dendritic puncta, respectively; cb, the PVD cell body; asterisks, gut autofluorescence. Anterior is at left and dorsal is up in all panels. Scale bar, 10 μm. (e,f) Quantification of axonal localization defects (e) and dendritic mislocalization defects (f) of RAB-3::mCherry and SAD-1::GFP, as in Figure 1; n > 30 worms per genotype. Error bars, s.e.p.

  5. UNC-104 is mislocalized to dendrites in unc-33 mutants.
    Figure 5: UNC-104 is mislocalized to dendrites in unc-33 mutants.

    (a,b) Immunostaining of endogenous UNC-104 in wild-type (a) and unc-33(mn407) (b) worms, with corresponding schematic diagrams. Yellow arrowheads, UNC-104 immunoreactivity in sensory dendrite regions; nr, nerve ring; vnc, ventral nerve cord. (c,d) Localization of UNC-104::GFP in PVD neurons of wild-type (c) and unc-33(mn407) (d) worms, with schematic diagrams. White arrowheads, UNC-104::GFP enrichment in PVD axons; yellow arrowheads, UNC-104::GFP in PVD dendrites. (e) Quantification of worms with detectable UNC-104::GFP fluorescence in PVD dendrites (n > 25 worms per genotype). Error bars, s.e.p. (f,g) Inferred UNC-104 activity in neurons of wild-type worms (f) and unc-33 mutants (g). Anterior is at left and dorsal is up in all panels. Scale bars, 10 μm.

  6. A sensory chemoreceptor protein is mislocalized to axons in unc-33 mutants.
    Figure 6: A sensory chemoreceptor protein is mislocalized to axons in unc-33 mutants.

    (a) Schematic diagram of AWB chemosensory neurons in the head. (b) Representative maximum projection fluorescence images showing ODR-10::GFP localization in AWB neurons of wild-type, unc-33, unc-104, and unc-104; unc-33 double mutant worms. Yellow arrowheads indicate ODR-10::GFP in axons. Anterior is at left and dorsal is up in all images. Scale bar, 10 μm. (c,d) Quantification of worms with ODR-10::GFP fluorescence in axons (n > 30 worms per genotype). Error bars, s.e.p.

  7. unc-44/ankyrin mutations disrupt polarized protein sorting and UNC-33L localization.
    Figure 7: unc-44/ankyrin mutations disrupt polarized protein sorting and UNC-33L localization.

    (a,b) Axonal (a) and dendritic (b) mislocalization defects of RAB-3::mCherry and SAD-1::GFP in unc-44 PVD neurons, as in Figure 1; n > 25 worms per genotype. (c) Immunostaining of endogenous UNC-104 in an unc-44(ky110) worm. Yellow arrowheads, UNC-104 immunoreactivity in sensory dendrite regions; nr, nerve ring; vnc, ventral nerve cord. Compare Figure 5a,b. (d) Representative maximum projection fluorescence image showing ODR-10::GFP localization in AWB neurons of an unc-44(ky110) worm. Yellow arrowhead, ODR-10::GFP in axons. Compare Figure 6b. (e,f) UNC-33L::GFP protein in PVD neurons of wild-type (e) and unc-44(ky110) (f) worms. Red brackets, region of UNC-33L enrichment in proximal axon; white arrowheads, axonal UNC-33L::GFP; yellow arrowheads, UNC-33L::GFP foci in dendrites and dendrite branches; asterisks, gut autofluorescence. (g) Quantification of worms with detectable UNC-33L::GFP in PVD axons and dendrite branches (n > 25 worms per genotype). All error bars indicate s.e.p. (h–j) Endogenous UNC-33L immunoreactivity in wild-type (h), unc-44(ky110) (i) and unc-33(ky880) (j) worms. Nerve ring (nr, top panels) and ventral nerve cord (vnc, bottom panels) are shown. Arrowheads indicate UNC-33L retained in neuronal cell bodies in unc-44 mutants. Anterior is at left and dorsal is up in all panels. Scale bars, 10 μm.

  8. Microtubule defects in unc-33 and unc-44 mutants.
    Figure 8: Microtubule defects in unc-33 and unc-44 mutants.

    (a,b) Representative maximum intensity projections of confocal images of α-tubulin immunoreactivity in head neurons of wild-type (a) and unc-33(ky880) (b) worms. White arrowheads, axon-rich nerve ring and ventral nerve cord; yellow arrowheads, distal sensory dendrites. unc-33(mn407) and unc-44(ky110) are shown in Supplementary Fig. 7, with quantification of immunostaining. (c,d) TBA-1::mCherry (α-tubulin) fluorescence in PVD neurons of representative wild-type (c) and unc-33(mn407) (d) worms. Scale bar in d, 10 μm (for a–d). (e–g) Representative kymographs of moving EBP-2::GFP puncta in axons (e), proximal dendrites (f) and distal dendrites (g) of head neurons of wild-type (top panel), unc-33(mn407) (middle) and unc-44(ky110) (bottom) worms. The cell body is to the right in all panels. Time runs top to bottom; arrowheads mark mutant puncta moving against the normal direction for that process. Bar graphs show the fractions of puncta moving anterogradely and retrogradely with respect to the cell body; arrows in key correspond to direction of movement in kymographs; MT, microtubule. Error bars, s.e.p. Numbers above each column denote the number of puncta counted in the corresponding categories.

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Author information

Affiliations

  1. Laboratory of Neural Circuits and Behavior, Howard Hughes Medical Institute, The Rockefeller University, New York, New York, USA.

    • Tapan A Maniar,
    • Miriam Kaplan &
    • Cornelia I Bargmann

  2. Department of Biology, Howard Hughes Medical Institute, Stanford University, California, USA.

    • George J Wang &
    • Kang Shen

  3. Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, Minnesota, USA.

    • Li Wei &
    • Jocelyn E Shaw

  4. National Centre for Biological Sciences – Tata Institute of Fundamental Research, Bangalore, India.

    • Sandhya P Koushika

Contributions

T.A.M. designed, conducted and interpreted most experiments and wrote the paper; M.K., G.J.W., K.S., L.W. and J.E.S. conducted and interpreted individual experiments with unc-33 and unc-44 mutants; S.P.K. generated the UNC-104 antibody and helped design transport experiments; C.I.B. designed and interpreted experiments and wrote the paper.

Competing financial interests

The authors declare no competing financial interests.

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Supplementary information

PDF files

  1. Supplementary Text and Figures (2M)

    Supplementary Figures 1–7

Movies

  1. Supplementary Video 1 (100K)

    Movement of EBP-2::GFP puncta in axons of wild type sensory neurons. Cell bodies are to the right. Displayed at 10x speed. Scale bar, 5 μm.

  2. Supplementary Video 2 (574K)

    Movement of EBP-2::GFP puncta in proximal dendrites of wild type sensory neurons. Distal dendrites are to the left; cell bodies are to the right. Displayed at 10x speed. Scale bar, 5 μm.

  3. Supplementary Video 3 (160K)

    Movement of EBP-2::GFP puncta in distal dendrites of wild type sensory neurons. Cilia are to the left; cell bodies are to the right (neither is visible). Displayed at 10x speed. Scale bar, 5 μm.

  4. Supplementary Video 4 (188K)

    Movement of EBP-2::GFP puncta in axons of unc-33(mn407) sensory neurons. Cell bodies are to the right. Displayed at 10x speed. Scale bar, 5 μm.

  5. Supplementary Video 5 (205K)

    Movement of EBP-2::GFP puncta in proximal dendrites of unc-33(mn407) sensory neurons. Distal dendrites are to the left; cell bodies are to the right. Displayed at 10x speed. Scale bar, 5 μm.

  6. Supplementary Video 6 (99K)

    Movement of EBP-2::GFP puncta in distal dendrites of unc-33(mn407) sensory neurons. Cilia are to the left; cell bodies are to the right (neither is visible). Displayed at 10x speed. Scale bar, 5 μm.

Additional data