A multi-protein receptor-ligand complex underlies combinatorial dendrite guidance choices in C. elegans
- Howard Hughes Medical Institute, Stanford University, United States
- University of California, Davis, United States
Figures
Figure 1 with 1 supplement
wy953 mutants show severe dendritic guidance defects.
(A) A cartoon showing the morphology of part of a PVD dendritic tree and its growth environment (adapted from Albeg et al., 2011). 1°: primary dendrites; 2°: secondary dendrites; 3°: tertiary …
Figure 1—figure supplement 1
PVD dendrites do not grow along all the SAX-7 positive loci.
Confocal images showing the subcellular localization of SAX-7::YFP expressed in the epidermis(left), the morphology of PVD dendritic arbors (middle) and overlay (right). Arrow heads: tertiary line …
Figure 2 with 1 supplement
Cloning and rescue of lect-2.
(A) Schematic of the LECT-2 protein with alleles indicated. (B–C) Confocal images showing the PVD dendritic arbors in lect-2(ok2617) mutants and lect-2(ok2617); wyTi3 [loxp -Plect-2>lect-2::SL2:: …
Figure 2—figure supplement 1
LECT-2 is homologous to human LECT2.
(A) Scores of identify and similarity between LECT-2 first half (1–168 amino acids), LECT-2 second half (169–333 amino acids) and human LECT2 (151 amino acids) using SIAS (http://imed.med.ucm.es/Tool…
Figure 3
lect-2 functions in the same genetic pathway as sax-7, mnr-1 and dma-1.
(A–H) Confocal images showing PVD dendritic arbors, body wall muscles and seam cells in (A) wild-type; (B) lect-2(wy953); (C) sax-7(nj48); (D) lect-2(wy953); sax-7(nj48); (E) mnr-1(wy758); (F) lect-2…
Figure 4 with 1 supplement
Transcriptional and translational expression patterns of lect-2.
(A) A confocal image showing the transcriptional expression pattern of lect-2 in an L4 stage animal. White arrows: body wall muscles. Green arrow: head neurons. Asterisks: auto fluorescence from the …
Figure 4—figure supplement 1
lect-2 is expressed in some ventral cord neurons.
Confocal images showing the morphology of PVD dendritic arbors and the mCherry expression driven by lect-2 endogenous promoter. Yellow arrow: ventral cord neurons. White arrow: a single body wall …
Figure 5
Body wall muscle-expressed LECT-2 is required for the patterning of 4° but not 2° or 3° dendrites.
(A–B) Confocal images showing the PVD dendritic arbors (labeled in green using transgene ser2prom3>myr-gfp) in lect-2(ok2617); wyTi3 [loxp-Plect-2>lect-2::SL2:: mcherry-loxp] without (A) or with (B) …
Figure 6 with 1 supplement
Muscle mosaic analysis.
(A–C) Confocal images showing PVD dendritic arbors, body wall muscles (arrow, labeled in magenta using transgene Phlh-1>lect-2::SL2::mcherry) and seam cells in a lect-2(ok2617) mutant animal which …
Figure 6—figure supplement 1
PVD patterning defect of lect-2 mutants is rescued by over-expressed LECT-2 secreted from multiple types of tissues.
(A–C) Quantifications showing the number of 2°, 3° and 4° dendrites in a 100 μm region anterior to the cell body of PVD neurons. One way ANOVA with the Tukey correction was used for statistical …
Figure 7
Seam cell-secreted LECT-2 could misguide dendrites onto seam cells.
(E) Confocal images showing PVD dendritic arbors, body wall muscles (labeled in magenta using transgene Pmyo-3>mcherry (for A, C–E) or Phlh-1> lect-2::SL2::mcherry (for C)) and seam cells. The inset …
Figure 8 with 2 supplements
LECT-2 interacts with SAX-7.
(A–C) Confocal images showing co-localization between LECT-2::GFP (green) and SAX-7::mCHERRY (magenta). Arrow heads: tertiary line along the border of outer body wall muscles; Arrows: quaternary …
Figure 8—figure supplement 1
Transcription of lect-2 is largely not affected by loss of sax-7.
(A–B) Confocal images showing the transcription reporter of lect-2 (Plect-2>mcherry) in wild-type and sax-7 (nj48) mutant animals. 28 and 24 animals were imaged for each genotype, respectively. No …
Figure 8—figure supplement 2
The last two FnIII domains of SAX-7 are sufficient to interact with LECT-2.
(A) Schematics showing that SAX-7 ECD CTF1 was generated through cleavage, likely in the third FnIII domain. (B) Western blot images showing co-IP results betweenLECT-2::FLAG and SAX-7 ECD CTF1::HA. …
Figure 9 with 1 supplement
LECT-2 forms a multi-protein complex with SAX-7, MNR-1 and DMA-1.
(A) A cartoon showing how the single-molecule pull-down (SiMPull) assays were performed. (B–J) Representive fluorescent images showing the results of SiMPull assays. Specific pull-down of LECT-2 …
Figure 9—figure supplement 1
Prey proteins are expressed at similar levels in different groups.
(A) C. elegans expressed LECT-2::GFP, SAX-7::mCherry, with or without co-expression of DMA-1::FLAG and MNR-1, were used in SiMPull. Lysates were applied to a chamber coated with anti-GFP or anti-RFP …
Figure 10 with 1 supplement
LECT-2 enhances the binding among SAX-7, MNR-1 and DMA-1 in vitro.
(A–B) Drosophila S2 cells expressing DMA-1::RFP and MNR-1::GFP, with or without co-expression of SAX-7::HA and LECT-2::FLAG, were used to detect protein interactions by SiMPull. Lysates were applied …
Figure 10—figure supplement 1
Adding LECT-2::FLAG increases the aggregation formation between DMA-1::RFP and SAX-7::GFP-MNR-1::GFP -expressing S2 cells.
(A–F) Fluorescent images showing the results of S2 cell aggregation assays at specific time points. Magenta cells: expressing DMA-1::RFP; Green cells: expressing SAX-7::GFP and MNR-1::GFP. –LECT-2: …
Additional files
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Supplementary file 1
Strains and plasmids used in this study.
(a) Strains used in this study. (b) Plasmids used in this study.
- https://doi.org/10.7554/eLife.18345.020
