Unleashed Actin Assembly in Capping Protein-Deficient B16-F1 Cells Enables Identification of Multiple Factors Contributing to Filopodium Formation
Background: Filopodia are dynamic, finger-like actin-filament bundles that overcome membrane tension by forces generated through actin polymerization at their tips to allow extension of these structures a few microns beyond the cell periphery. Actin assembly of these protrusions is regulated by acce...
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| Format: | Article |
| Language: | English |
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MDPI AG
2023-03-01
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| Series: | Cells |
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| Online Access: | https://www.mdpi.com/2073-4409/12/6/890 |
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| author | Jens Ingo Hein Jonas Scholz Sarah Körber Thomas Kaufmann Jan Faix |
| author_facet | Jens Ingo Hein Jonas Scholz Sarah Körber Thomas Kaufmann Jan Faix |
| author_sort | Jens Ingo Hein |
| collection | DOAJ |
| description | Background: Filopodia are dynamic, finger-like actin-filament bundles that overcome membrane tension by forces generated through actin polymerization at their tips to allow extension of these structures a few microns beyond the cell periphery. Actin assembly of these protrusions is regulated by accessory proteins including heterodimeric capping protein (CP) or Ena/VASP actin polymerases to either terminate or promote filament growth. Accordingly, the depletion of CP in B16-F1 melanoma cells was previously shown to cause an explosive formation of filopodia. In Ena/VASP-deficient cells, CP depletion appeared to result in ruffling instead of inducing filopodia, implying that Ena/VASP proteins are absolutely essential for filopodia formation. However, this hypothesis was not yet experimentally confirmed. Methods: Here, we used B16-F1 cells and CRISPR/Cas9 technology to eliminate CP either alone or in combination with Ena/VASP or other factors residing at filopodia tips, followed by quantifications of filopodia length and number. Results: Unexpectedly, we find massive formations of filopodia even in the absence of CP and Ena/VASP proteins. Notably, combined inactivation of Ena/VASP, unconventional myosin-X and the formin FMNL3 was required to markedly impair filopodia formation in CP-deficient cells. Conclusions: Taken together, our results reveal that, besides Ena/VASP proteins, numerous other factors contribute to filopodia formation. |
| first_indexed | 2024-03-11T06:47:22Z |
| format | Article |
| id | doaj.art-1e315a70a21c4267a747929baa5db391 |
| institution | Directory Open Access Journal |
| issn | 2073-4409 |
| language | English |
| last_indexed | 2024-03-11T06:47:22Z |
| publishDate | 2023-03-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Cells |
| spelling | doaj.art-1e315a70a21c4267a747929baa5db3912023-11-17T10:13:18ZengMDPI AGCells2073-44092023-03-0112689010.3390/cells12060890Unleashed Actin Assembly in Capping Protein-Deficient B16-F1 Cells Enables Identification of Multiple Factors Contributing to Filopodium FormationJens Ingo Hein0Jonas Scholz1Sarah Körber2Thomas Kaufmann3Jan Faix4Institute for Biophysical Chemistry, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, GermanyInstitute for Biophysical Chemistry, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, GermanyInstitute for Biophysical Chemistry, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, GermanyInstitute for Biophysical Chemistry, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, GermanyInstitute for Biophysical Chemistry, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, GermanyBackground: Filopodia are dynamic, finger-like actin-filament bundles that overcome membrane tension by forces generated through actin polymerization at their tips to allow extension of these structures a few microns beyond the cell periphery. Actin assembly of these protrusions is regulated by accessory proteins including heterodimeric capping protein (CP) or Ena/VASP actin polymerases to either terminate or promote filament growth. Accordingly, the depletion of CP in B16-F1 melanoma cells was previously shown to cause an explosive formation of filopodia. In Ena/VASP-deficient cells, CP depletion appeared to result in ruffling instead of inducing filopodia, implying that Ena/VASP proteins are absolutely essential for filopodia formation. However, this hypothesis was not yet experimentally confirmed. Methods: Here, we used B16-F1 cells and CRISPR/Cas9 technology to eliminate CP either alone or in combination with Ena/VASP or other factors residing at filopodia tips, followed by quantifications of filopodia length and number. Results: Unexpectedly, we find massive formations of filopodia even in the absence of CP and Ena/VASP proteins. Notably, combined inactivation of Ena/VASP, unconventional myosin-X and the formin FMNL3 was required to markedly impair filopodia formation in CP-deficient cells. Conclusions: Taken together, our results reveal that, besides Ena/VASP proteins, numerous other factors contribute to filopodia formation.https://www.mdpi.com/2073-4409/12/6/890filopodiacapping proteinEna/VASP proteinsmyosin-XmDia2FMNL2 |
| spellingShingle | Jens Ingo Hein Jonas Scholz Sarah Körber Thomas Kaufmann Jan Faix Unleashed Actin Assembly in Capping Protein-Deficient B16-F1 Cells Enables Identification of Multiple Factors Contributing to Filopodium Formation Cells filopodia capping protein Ena/VASP proteins myosin-X mDia2 FMNL2 |
| title | Unleashed Actin Assembly in Capping Protein-Deficient B16-F1 Cells Enables Identification of Multiple Factors Contributing to Filopodium Formation |
| title_full | Unleashed Actin Assembly in Capping Protein-Deficient B16-F1 Cells Enables Identification of Multiple Factors Contributing to Filopodium Formation |
| title_fullStr | Unleashed Actin Assembly in Capping Protein-Deficient B16-F1 Cells Enables Identification of Multiple Factors Contributing to Filopodium Formation |
| title_full_unstemmed | Unleashed Actin Assembly in Capping Protein-Deficient B16-F1 Cells Enables Identification of Multiple Factors Contributing to Filopodium Formation |
| title_short | Unleashed Actin Assembly in Capping Protein-Deficient B16-F1 Cells Enables Identification of Multiple Factors Contributing to Filopodium Formation |
| title_sort | unleashed actin assembly in capping protein deficient b16 f1 cells enables identification of multiple factors contributing to filopodium formation |
| topic | filopodia capping protein Ena/VASP proteins myosin-X mDia2 FMNL2 |
| url | https://www.mdpi.com/2073-4409/12/6/890 |
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