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Backgrounds <p>The cellular slime mold <i>Dictyostelium discoideum</i> is a widely used model system for studying basic processes in cell and developmental biology. While genetic tools, such as targeted gene disruption by homologous recombination and genome editing using CRISPR/Cas9, are well-established in <i>D. discoideum</i>, efficient methods for conditional loss-of-function studies are limited. Here, we developed a nanobody-based degron system for <i>D. discoideum</i> based on ALFA-tagged protein recruitment to the Skp1-Cullin-F-box (SCF) complex.</p> Results <p>ALFA-tagged Histone H1 was efficiently degraded by expressing anti-ALFA nanobody (NbALFA) fused to the <i>D. discoideum</i> FbxD F-box domain (‘dictyGrad-ALFA’). Cell type-specific targeting was achieved by expressing dictyGrad-ALFA under prestalk- and prespore-specific gene promoters. Furthermore, targeting of adenylyl cyclase A (ACA) resulted in the expected aggregation-deficient phenotype, validating the efficacy of dictyGrad-ALFA-mediated protein depletion. Cell type-specific ACA degradation delayed development but eventually resulted in normal fruiting bodies. Our ALFA-tag approach was further used for conditional knockdown in combination with the auxin-inducible degron 2 (AID2) system, which relies on indole-3-acetic acid (IAA)-dependent binding between NbALFA-mAID and a OsTIR-F-box-Skp1A fusion protein. We obtained efficient IAA-induced degradation in prestalk cells; however, efficiency was low in other cell types.</p> Conclusions <p>Together, these systems pave the way for conditional and cell type-specific protein degradation in <i>D. discoideum</i>, enabling functional analyses of genes essential for growth and development.</p>

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A nanobody-based degron system for targeted protein knockdown in Dictyostelium discoideum

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年2月19日

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  • Hidenori Hashimura,
  • Shoko Fujishiro,
  • Nao Shimada,
  • Tomoko Adachi,
  • Toyoko Sugita,
  • Satoshi Kuwana,
  • Satoshi Sawai

摘要

Backgrounds

The cellular slime mold Dictyostelium discoideum is a widely used model system for studying basic processes in cell and developmental biology. While genetic tools, such as targeted gene disruption by homologous recombination and genome editing using CRISPR/Cas9, are well-established in D. discoideum, efficient methods for conditional loss-of-function studies are limited. Here, we developed a nanobody-based degron system for D. discoideum based on ALFA-tagged protein recruitment to the Skp1-Cullin-F-box (SCF) complex.

Results

ALFA-tagged Histone H1 was efficiently degraded by expressing anti-ALFA nanobody (NbALFA) fused to the D. discoideum FbxD F-box domain (‘dictyGrad-ALFA’). Cell type-specific targeting was achieved by expressing dictyGrad-ALFA under prestalk- and prespore-specific gene promoters. Furthermore, targeting of adenylyl cyclase A (ACA) resulted in the expected aggregation-deficient phenotype, validating the efficacy of dictyGrad-ALFA-mediated protein depletion. Cell type-specific ACA degradation delayed development but eventually resulted in normal fruiting bodies. Our ALFA-tag approach was further used for conditional knockdown in combination with the auxin-inducible degron 2 (AID2) system, which relies on indole-3-acetic acid (IAA)-dependent binding between NbALFA-mAID and a OsTIR-F-box-Skp1A fusion protein. We obtained efficient IAA-induced degradation in prestalk cells; however, efficiency was low in other cell types.

Conclusions

Together, these systems pave the way for conditional and cell type-specific protein degradation in D. discoideum, enabling functional analyses of genes essential for growth and development.