The KASH protein UNC-83 differentially regulates kinesin-1 activity to control developmental stage-specific nuclear migration

Abstract

Nuclear migration plays a fundamental role in development, requiring precise spatiotemporal control of bidirectional movement through dynein and kinesin motors. Here, we uncover a differential isoform-dependent mechanism for developmental regulation of nuclear migration directionality. The nuclear envelope Klarsicht/ANC-1/Syne homology (KASH) protein UNC-83 in Caenorhabditis elegans exists in multiple isoforms that differentially control motor activity to achieve tissue-specific nuclear positioning. The shorter UNC-83c isoform promotes kinesin-1-dependent nuclear movement in embryonic hyp7 precursors, while longer UNC-83a/b isoforms facilitate dynein-mediated nuclear migration in larval P cells. We demonstrate that the UNC-83a-specific N-terminal domain functions as a kinesin-1 inhibitory module by directly binding the kinesin heavy chain (UNC-116). This interaction prevents kinesin-1 activation and reduces the protein's affinity for kinesin light chain (KLC-2), allowing for dynein-mediated transport. By contrast, UNC-83c exhibits high-affinity binding to KLC-2, promoting kinesin-1 activation for plus-end-directed movement. AlphaFold structural predictions reveal that UNC-83 contains five spectrin-like repeats, with two located within the inhibitory N-terminal domain. Genetic analysis demonstrates that these spectrin-like repeats are essential for dynein-dependent P cell nuclear migration but dispensable for kinesin-1-dependent hyp7 migration. This isoform-specific inhibition, combined with differential affinity for KLC-2, establishes a mechanism for achieving directional control of nuclear positioning during development. Together, these interdisciplinary studies reveal how alternative isoforms of cargo adaptors can generate developmental stage-specific regulation of motor activity.

Keywords: C. elegans; KASH proteins; LINC complex; dynein; kinesin-1; nuclear migration.

Figure 1:. Different isoforms of UNC-83 mediate nuclear migration throughout development.

A) Schematic showing the kinesin-1-mediated migration of embryonic hypodermal hyp7 nuclei (cyan) across the dorsal midline (gray) towards the plus-ends of microtubules (green). B) Schematic illustrating the dynein-driven migration of larval P-cell nuclei (cyan) towards the ventral cord (gray), where the minus-ends of microtubules are positioned. C) Model of the hypothesis tested in this study. The short isoform UNC-83c activates kinesin-1 and/or inhibit dynein, making kinesin-1 the main motor driving embryonic hyp7 nuclear migration. The longer isoforms UNC-83a/b activate dynein and/or inhibit kinesin-1, making dynein the main driver of larval P-cell nuclear migration. D) AlphaFold3 predicted structure of the long isoform, UNC-83a. C-D) Spectrin-like repeats (SRs) SR1 and SR2 (unique to UNC-83a/b) are in magenta; SR3, SR4 and SR5 (common to all isoforms) are in dark blue. The transmembrane domain (TM) is shown in dark gray. The KASH peptide is shown in cyan. The EWD motif is highlighted in red (see Figure S1 for confidence and error scores of the predicted structure).

Figure 2:. The long UNC-83a isoform binds KLC-2 with significantly lower affinity than the short UNC-83c isoform.

A) AlphaFold3-predicted interaction of UNC-83c’s EWD motif (red) with the TPR domain (green) of the kinesin light chain, KLC-2. B) Coomassie-stained protein gels of purified UNC-83a, UNC-83c and KLC-2 recombinant proteins. C) Schematic of the GST-KLC-2 construct (GST tag in gray, coiled-coil domain in dark green, and TPR domain in light green) and a Gaussian fit of its measured molecular weight in solution (30 nM; monomer = 92 ± 19.8 kDa, 72% peak population; dimer = 177 ± 59 kDa, 21% peak) as determined by mass photometry. D) Schematics and molecular weight distributions of UNC-83c-mScarlet (30 nM; monomer = 99 ± 47 kDa, 95% peak) and UNC-83a-mScarlet (30 nM; monomer = 130 ± 62 kDa, 96% peak). SR1 and SR2 (unique to UNC-83a/b) are in magenta; SR3, SR4 and SR5 (common to all isoforms) are in dark blue; mScarlet-I tag in fuchsia, and Strep-II tag in dark gray. E) Top, schematics showing the interaction between UNC-83c and KLC-2 (on the left), and the lack of interaction between UNC-83a and KLC-2 (on the right). Bottom, molecular weight distributions of KLC-2 preincubated with UNC-83c (30 nM; one UNC-83c monomer + one KLC-2 monomer = 174 ± 134 kDa, 97% peak) and UNC-83a (30 nM; one UNC-83a monomer = 138 ± 62 kDa, 80% peak). F) Binding curves of UNC-83a and UNC-83c to KLC-2, showing an affinity of 0.15 μM for UNC-83c and >1.0 μM for UNC-83a. The bait, GST-KLC-2, was diluted to 1 μM and immobilized onto the biosensors. Both UNC-83a and UNC-83c were tested at concentrations of 1.0, 0.667, 0.444, 0.296, 0.197, 0.132, 0.088 μM.

Figure 3:. Abnormal presence of the long unc-83a isoform in embryonic hyp7 precursors disrupts kinesin-1-dependent nuclear migration, while expression of the short unc-83c isoform in larval P cells disrupts dynein-mediated nuclear migration.

A) Schematic of the unc-83(yc108[UNC-83a]) mutant, expressing the unc-83a isoform under control of the unc-83c promoter at the endogenous unc-83c locus. B) UNC-83 (shown in red) in unc-83(yc108[UNC-83a]) is localized to the nuclear envelope in a comma-stage embryo. Dorsal is up and anterior is left. Arrows point to the hyp7 nuclei. C) Quantification of hyp7 nuclear migration defects in wild type, unc-83(yc108[UNC-83a]), and unc-83(e1408) null animals via counting the number of mislocalized (at the dorsal cord) nuclei. Each point represents the total number of abnormally located hyp7 nuclei per animal; n = 40 for each strain. D) Lateral views of wild type and unc-83(yc108[UNC-83a]) L4 larva expressing hypodermal nuclear GFP. Dashed yellow lines mark the dorsal cord of the animal. Mislocalized nuclei (arrows) indicate migration failure. E) Quantification of dynein-mediated larval P-cell nuclear migration defects in wild type, mutant unc-83(yc108[UNC-83a]), and unc-83(e1408) null animals via counting number of missing GABA neurons at 25°C. Each point represents the number of missing GABA neurons per animal; n = 40 for each strain. F) Lateral views of wild type and unc-83(yc108[UNC-83a]) L4 larva expressing a GABA neuron nuclear marker (unc-47::gfp). Numbers indicate a total of 19 GABA neurons. G) Quantification of larval P-cell nuclear migration defects in wild type, unc-83(e1408) null, and unc-83c overexpression animals (unc-83(ycEx301[phlh3::unc-83c(+)]) and unc-83(ycEx302[phlh3::unc-83c(+)])) via counting number of missing GABA neurons at 25°C. Both transgenic lines show mild but statistically significant defects in P-cell nuclear migration, with 0.45 ± 0.20 and 0.75 ± 0.33 missing GABA neurons compared to 0.1 ± 0.09 missing GABA neurons in wild type. Each point represents the number of missing GABA neurons per animal; n = 40 for each strain. H) Lateral view of unc-83(ycEx302[phlh3::unc-83c(+)]) L4 larvae expressing unc-47::gfp. Numbers indicate a total of 16 GABA neurons in larva. For all plots: Means with 95% CI are shown in error bars. Unpaired student t-tests were performed on the indicated comparisons; ns means not significant, p>0.05; * p<0.05; **** p<0.0001. For images: Scale bars = 10 μm for B), and 42.2 μm for D), F) and H).

Figure 4:. Spectrin-like repeats in the N-terminal domain of UNC-83a are necessary for dynein-dependent nuclear migration.

A) Schematics of the unc-83 N-terminal domain deletion mutants. B) Immunofluorescence staining shows that the deletion mutants unc-83a(Δ234–305), unc-83a(Δ58–166), unc-83a(Δ58–233), and unc-83a(Δ167–233) have a normal UNC-83 (red) localization pattern at the nuclear envelope of hyp7 precursor cell nuclei in comma-stage embryos. White arrows indicate nuclei with UNC-83 localization. Dorsal is up and anterior is left. C) Quantification of larval P-cell nuclear migration defects in the indicated strains at 25°C (See Figure S2 for the P-cell nuclear migration defects of the indicated strains at 20°C and 15°C.). Each point represents the number of missing GABA neurons per animal; n = 40 for each strain. Wild type and unc-83(e1408) data are the same as shown in Figure 3. D) Lateral views of unc-83a(Δ234–305) and unc-83a(Δ58–233) L4 larvae expressing unc-47::gfp. Numbers indicate total GABA neurons. E) Quantification of hyp7 nuclear migration defects in the indicated strains. Each point represents the total number of abnormally located hyp7 nuclei per animal; n = 40 for each strain. Wild type and unc-83(e1408) data are the same as shown in Figure 3. F) Lateral views of unc-83a(Δ234–305) and unc-83a(Δ58–233) L4 larvae expressing hypodermal nuclear GFP. Dashed yellow lines mark the dorsal cord of the animals. For all plots: Means with 95% CI are shown in error bars. Unpaired student t-tests were performed on the indicated comparisons; ns means not significant, p>0.05; * p<0.05; **** p<0.0001. For images: Scale bars = 10 μm for B) and 42.2 μm for D) and F). See Figure S2 for more details.

Figure 5:. A conserved enhancer controls cell-type specific expression of unc-83c in embryonic hypodermal precursors.

A) Schematics of the unc-83 locus in wild type, and enhancer deletion mutants (unc-83(yc121) and unc-83(yc122)). The 870bp enhancer region (see Figure S3 for sequences) is highlighted in red. Exons are shown as blue boxes. The start codon for unc-83a in exon 1, unc-83b in exon 3, and unc-83c at the end of exon 8 are indicated. The sizes of introns and exons are according to the scale as indicated (scale bar=1 kb). (See Figure S3 for the sequence alignment). B) Quantification of hypodermal hyp7 nuclear migration defects in the indicated strains. Each point represents the total number of abnormally located hyp7 nuclei per animal; n = 40 for each strain. Wild type and unc-83(e1408) data are the same as shown in Figure 3. C) Lateral view of unc-83(yc121) L4 larva expressing hypodermal nuclear GFP. Dashed yellow line mark the dorsal of the animal. Arrows indicate mislocalized hypodermal nuclei, representing failed nuclear migrations. D) Quantification of larval P-cell nuclear migration defects in the indicated strains at 25°C. Each point represents the number of missing GABA neurons per animal; n = 40 for each strain. Wild type and unc-83(e1408) data are the same as shown in Figure 3. E) Lateral view of unc-83(yc121) L4 larva expressing unc-47::gfp. Numbers indicate a total number of 18 GABA neurons. F-G) C. elegans pre-comma stage embryos expressing unc-83 870bp enhancer region driving nls::gfp. F) Expression of the 870 bp enhancer region (green) in pre-comma stage embryo, showing hyp7 precursor-specific expression. For all plots: Means with 95% CI are shown in error bars. Unpaired student t-tests were performed on the indicated comparisons; ns means not significant, p>0.05; * p<0.05; **** p<0.0001. For images: Scale bars = 42.2 μm for C) and E) and 10 μm for F) and G).

Figure 6:. The UNC-83a N-terminal domain inhibits kinesin-1 activity in vitro.

A) Schematics of the indicated recombinant protein constructs. The coiled-coil domains of UNC-116 and KLC-2 are indicated as ‘CC’. UNC-116 is co-expressed with KLC-2. The co-expression leads to the formation of kinesin-1 heterotetramer that is expected to be 362 kDa. B) Coomassie- stained gels of purified UNC-83a^N-term and UNC-116-KLC-2 (kinesin-1) recombinant proteins. Arrows indicate the bands for the different proteins. C) Representative TIRF images showing the recruitment of kinesin-1 (green) in the rigor state (with 2 mM AMP-PNP) onto microtubules (blue) and the effects of the indicated UNC-83 constructs (magenta). Scale bar = 2.5 μm. D) Quantification of microtubule-bound kinesin-1 fluorescence intensity (normalized to the kinesin-1 signal when no UNC-83 constructs were added) when kinesin-1 is preincubated with indicated UNC-83 constructs. N = 3, n = 30 microtubules analyzed for each condition. E) Representative kymographs of microtubule gliding with kinesin-1 alone (See Video S1), kinesin-1 + UNC-83c (See Video S2), kinesin-1 + UNC-83a (See Video S3), and kinesin-1 + UNC-83a^N-term (See Video S4). Scale bars: 20 s (vertical) and 5 μm (horizontal). F) Quantification of microtubule gliding velocities for the indicated conditions. N=2, n = 25 microtubules analyzed for each condition. The average velocities are: 1,064 ± 35 nm/s (kinesin-1), 1,150 ± 149 nm/s (kinesin-1 + UNC-83c), 1,309 ± 82 nm/s (kinesin-1 + UNC-83a), 187 ± 533 nm/s (kinesin-1 + UNC-83a^N-term).

Figure 7:. The UNC-83a N-terminal domain directly binds kinesin heavy chain.

A) A Coomassie-stained gel showing purified UNC-116-sfGFP. B) A schematic of the UNC-116-sfGFP recombinant protein and a Gaussian fit of its measured molecular weight in solution by mass photometry (30 nM; monomer = 109 ± 19.5 kDa, 13% peak; dimer = 246 ± 44 kDa; 82% peak). Coiled-coil domains indicated as ‘CC’. C) A Gaussian fit of UNC-116-KLC-2 (kinesin-1) recombinant protein’s measured molecular weight in solution by mass photometry (30 nM; tetramer = 360 ± 69 kDa, 88% peak). D-D”) A schematic of the UNC-83a-mScarlet recombinant protein and a Gaussian fit of its measured molecular weight in solution alone (30 nM; monomer = 130 ± 62 kDa; 96% peak) (D), when preincubated with UNC-116 (30 nM; one UNC-83a monomer + one UNC-116 dimer = 391 ± 153 kDa, 99% peak) (D’), and when preincubated with UNC-116-KLC-2 (kinesin-1) (30 nM; one UNC-83a monomer + one kinesin-1 tetramer = 531 ± 272 kDa, 97% peak) (D”). E-E’) A schematic of the UNC-83a^N-term-mScarlet recombinant protein and a Gaussian fit of its measured molecular weight in solution alone (30 nM; dimer = 120 ± 23 kDa, 100% peak) (E) or when preincubated with UNC-116 (30 nM; one UNC-83a^N-term monomer + one UNC-116 dimer = 307 ± 89 kDa, 99% peak) (E’) or when preincubated with UNC-116-KLC-2 (kinesin-1) (30 nM; 398 ± 132 kDa, 72% peak) (E”). F-F”) A schematic of the UNC-83c-mScarlet recombinant protein and a Gaussian fit of its measured molecular weight in solution alone (30 nM; monomer = 108 ± 41 kDa, 100% peak) (F), when preincubated with UNC-116 (30 nM; 103 ± 21 kDa, 30%; 245 ± 82 kDa, 70% peak) (F’), and when preincubated with UNC-116-KLC-2 (kinesin-1) (30 nM; 128 ± 41 kDa, 24% peak; 360 ± 110 kDa, 72% peak) (F”).