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

Maniar TA, Kaplan M, Wang GJ, Shen K, Wei L, Shaw JE, Koushika SP, Bargmann CI - Nat. Neurosci. (2011)

Bottom Line: 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.We suggest that UNC-44 and UNC-33 direct polarized sorting through their global effects on neuronal microtubule organization.

View Article: PubMed Central - PubMed

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

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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UNC-33L is enriched in PVD axons(a) UNC-33L immunoreactivity in nerve ring of wild-type animal.(b) Biologically active UNC-33L::GFP protein expressed from a pan-neuronal promoter in wild type animal, 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::GFP proteins in wild type PVD neurons, with schematic diagrams at right. Red brackets indicate region of UNC-33L enrichment in axon, and arrowheads show expression in ventral nerve cord. Black brackets indicate proximal segment of PVD axon used for comparing fluorescence intensities in (f).(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 indicate s.e.m. *** p < 0.001 according to the Bonferroni t-test, ns, not significant. Anterior is at left and dorsal is up in all panels. Scale bars, 10 μm.
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Figure 3: UNC-33L is enriched in PVD axons(a) UNC-33L immunoreactivity in nerve ring of wild-type animal.(b) Biologically active UNC-33L::GFP protein expressed from a pan-neuronal promoter in wild type animal, 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::GFP proteins in wild type PVD neurons, with schematic diagrams at right. Red brackets indicate region of UNC-33L enrichment in axon, and arrowheads show expression in ventral nerve cord. Black brackets indicate proximal segment of PVD axon used for comparing fluorescence intensities in (f).(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 indicate s.e.m. *** p < 0.001 according to the Bonferroni t-test, ns, not significant. Anterior is at left and dorsal is up in all panels. Scale bars, 10 μm.

Mentions: UNC-33L has been reported to be enriched in nerve ring axons 27. We generated a new polyclonal antibody against UNC-33L and confirmed its axonal localization in the nerve ring and ventral cord, and its absence from sensory dendrites (Fig. 3a). A similar pattern of axonal enrichment was observed with an internally-tagged UNC-33L::GFP protein that rescued unc-33 locomotion and protein localization defects (Fig. 3b). UNC-33L::GFP expressed under the des-2 promoter labeled PVD axons consistently, but was minimally expressed in primary PVD dendrites and was undetectable in dendrite branches (Fig. 3c). Within the PVD axon, UNC-33L::GFP was enriched in a segment of the axon near, but not adjacent to, the PVD cell body (Fig. 3c). When expressed separately, the unique N-terminal UNC-33L domain was enriched in axons, but not in this small axon segment, whereas the smaller UNC-33S protein was randomly distributed to axons and dendrites (Fig. 3d-f). Enrichment of UNC-33L::GFP in axonal segments near the cell body was also observed in FLP and AWC neurons (data not shown).


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

Maniar TA, Kaplan M, Wang GJ, Shen K, Wei L, Shaw JE, Koushika SP, Bargmann CI - Nat. Neurosci. (2011)

UNC-33L is enriched in PVD axons(a) UNC-33L immunoreactivity in nerve ring of wild-type animal.(b) Biologically active UNC-33L::GFP protein expressed from a pan-neuronal promoter in wild type animal, 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::GFP proteins in wild type PVD neurons, with schematic diagrams at right. Red brackets indicate region of UNC-33L enrichment in axon, and arrowheads show expression in ventral nerve cord. Black brackets indicate proximal segment of PVD axon used for comparing fluorescence intensities in (f).(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 indicate s.e.m. *** p < 0.001 according to the Bonferroni t-test, ns, not significant. Anterior is at left and dorsal is up in all panels. Scale bars, 10 μm.
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Related In: Results  -  Collection

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Figure 3: UNC-33L is enriched in PVD axons(a) UNC-33L immunoreactivity in nerve ring of wild-type animal.(b) Biologically active UNC-33L::GFP protein expressed from a pan-neuronal promoter in wild type animal, 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::GFP proteins in wild type PVD neurons, with schematic diagrams at right. Red brackets indicate region of UNC-33L enrichment in axon, and arrowheads show expression in ventral nerve cord. Black brackets indicate proximal segment of PVD axon used for comparing fluorescence intensities in (f).(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 indicate s.e.m. *** p < 0.001 according to the Bonferroni t-test, ns, not significant. Anterior is at left and dorsal is up in all panels. Scale bars, 10 μm.
Mentions: UNC-33L has been reported to be enriched in nerve ring axons 27. We generated a new polyclonal antibody against UNC-33L and confirmed its axonal localization in the nerve ring and ventral cord, and its absence from sensory dendrites (Fig. 3a). A similar pattern of axonal enrichment was observed with an internally-tagged UNC-33L::GFP protein that rescued unc-33 locomotion and protein localization defects (Fig. 3b). UNC-33L::GFP expressed under the des-2 promoter labeled PVD axons consistently, but was minimally expressed in primary PVD dendrites and was undetectable in dendrite branches (Fig. 3c). Within the PVD axon, UNC-33L::GFP was enriched in a segment of the axon near, but not adjacent to, the PVD cell body (Fig. 3c). When expressed separately, the unique N-terminal UNC-33L domain was enriched in axons, but not in this small axon segment, whereas the smaller UNC-33S protein was randomly distributed to axons and dendrites (Fig. 3d-f). Enrichment of UNC-33L::GFP in axonal segments near the cell body was also observed in FLP and AWC neurons (data not shown).

Bottom Line: 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.We suggest that UNC-44 and UNC-33 direct polarized sorting through their global effects on neuronal microtubule organization.

View Article: PubMed Central - PubMed

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

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.

Show MeSH
Related in: MedlinePlus