RESUMEN
The actin cytoskeleton is the primary driver of cellular adhesion and mechanosensing due to its ability to generate force and sense the stiffness of the environment. At the cell's leading edge, severing of the protruding Arp2/3 actin network generates a specific actin/tropomyosin (Tpm) filament population that controls lamellipodial persistence. The interaction between these filaments and adhesion to the environment is unknown. Using cellular cryo-electron tomography we resolve the ultrastructure of the Tpm/actin copolymers and show that they specifically anchor to nascent adhesions and are essential for focal adhesion assembly. Re-expression of Tpm1.8/1.9 in transformed and cancer cells is sufficient to restore cell-substrate adhesions. We demonstrate that knock-out of Tpm1.8/1.9 disrupts the formation of dorsal actin bundles, hindering the recruitment of α-actinin and non-muscle myosin IIa, critical mechanosensors. This loss causes a force-generation and proliferation defect that is notably reversed when cells are grown on soft surfaces. We conclude that Tpm1.8/1.9 suppress the metastatic phenotype, which may explain why transformed cells naturally downregulate this Tpm subset during malignant transformation.
Asunto(s)
Neoplasias , Tropomiosina , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Proliferación Celular , Seudópodos/metabolismo , Tropomiosina/metabolismoRESUMEN
Genetic tags allow rapid localization of tagged proteins in cells and tissues. APEX, an ascorbate peroxidase, has proven to be one of the most versatile and robust genetic tags for ultrastructural localization by electron microscopy (EM). Here, we describe a simple method, APEX-Gold, which converts the diffuse oxidized diaminobenzidine reaction product of APEX into a silver/gold particle akin to that used for immunogold labelling. The method increases the signal-to-noise ratio for EM detection, providing unambiguous detection of the tagged protein, and creates a readily quantifiable particulate signal. We demonstrate the wide applicability of this method for detection of membrane proteins, cytoplasmic proteins, and cytoskeletal proteins. The method can be combined with different EM techniques including fast freezing and freeze substitution, focussed ion beam scanning EM, and electron tomography. Quantitation of expressed APEX-fusion proteins is achievable using membrane vesicles generated by a cell-free expression system. These membrane vesicles possess a defined quantum of signal, which can act as an internal standard for determination of the absolute density of expressed APEX-fusion proteins. Detection of fusion proteins expressed at low levels in cells from CRISPR-edited mice demonstrates the high sensitivity of the APEX-Gold method.