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Filopodia formation and endosome clustering induced by mutant plus-end-directed myosin VI
Thomas A Masters1, Folma Buss1
1Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 0XY, United Kingdom fb207@cam.ac.uk tam55@cam.ac.uk.
Abstract:
Myosin VI (MYO6) is the only myosin known to move toward the minus end of actin filaments. It has roles in numerous cellular processes, including maintenance of stereocilia structure, endocytosis, and autophagosome maturation. However, the functional necessity of minus-end-directed movement along actin is unclear as the underlying architecture of the local actin network is often unknown. To address this question, we engineered a mutant of MYO6, MYO6+, which undergoes plus-end-directed movement while retaining physiological cargo interactions in the tail. Expression of this mutant motor in HeLa cells led to a dramatic reorganization of cortical actin filaments and the formation of actin-rich filopodia. MYO6 is present on peripheral adaptor protein, phosphotyrosine interacting with PH domain and leucine zipper 1 (APPL1) signaling endosomes and MYO6+ expression causes a dramatic relocalization and clustering of this endocytic compartment in the cell cortex. MYO6+ and its adaptor GAIP interacting protein, C terminus (GIPC) accumulate at the tips of these filopodia, while APPL1 endosomes accumulate at the base. A combination of MYO6+ mutagenesis and siRNA-mediated depletion of MYO6 binding partners demonstrates that motor activity and binding to endosomal membranes mediated by GIPC and PI(4,5)P2 are crucial for filopodia formation. A similar reorganization of actin is induced by a constitutive dimer of MYO6+, indicating that multimerization of MYO6 on endosomes through binding to GIPC is required for this cellular activity and regulation of actin network structure. This unique engineered MYO6+ offers insights into both filopodia formation and MYO6 motor function at endosomes and at the plasma membrane.
Insights
Engineered Myosin VI (MYO6) mutant (MYO6+) drives plus-end movement, reorganizing actin networks and forming filopodia. This reveals MYO6
Area of Science:
- Cell Biology
- Molecular Motors
- Cytoskeleton Dynamics
Background:
- Myosin VI (MYO6) is unique for its minus-end-directed actin movement.
- Its role in cellular processes like endocytosis and actin network organization is not fully understood.
- The local actin architecture influencing MYO6 function remains largely unknown.
Purpose of the Study:
- To investigate the functional necessity of MYO6's minus-end-directed movement.
- To engineer a MYO6 mutant (MYO6+) with plus-end-directed motility.
- To explore the impact of altered MYO6 motor activity on cellular actin organization and endosomes.
Main Methods:
- Engineering of a plus-end-directed MYO6 mutant (MYO6+).
- Expression of MYO6+ in HeLa cells to observe actin and endosome dynamics.
- Mutagenesis of MYO6+ and siRNA-mediated depletion of binding partners.
- Analysis of APPL1 endosome localization and filopodia formation.
Main Results:
- MYO6+ expression induced significant cortical actin reorganization and filopodia formation.
- APPL1-containing endosomes relocalized and clustered at the cell cortex.
- MYO6+ and GIPC accumulated at filopodia tips, while APPL1 endosomes localized at the base.
- Motor activity and GIPC/PI(4,5)P2 binding were essential for filopodia induction.
- Constitutive dimerization of MYO6+ mimicked the actin reorganization, highlighting multimerization importance.
Conclusions:
- Minus-end-directed movement is not essential for MYO6's role in actin organization and filopodia formation.
- Engineered plus-end-directed MYO6 (MYO6+) reveals motor activity and specific binding interactions are key for actin network regulation.
- MYO6 multimerization on endosomes via GIPC is critical for inducing actin structure changes.
- This study provides insights into filopodia formation and MYO6 function at endosomes and the plasma membrane.
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