Myosin-X and talin modulate integrin activity at filopodia tips.
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ABSTRACT:
SUBMITTER: Miihkinen M
PROVIDER: S-EPMC8456781 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Myosin-X (Myo10) is an unconventional myosin with MyTH4-FERM domains that is best known for its striking localization to the tips of filopodia and its ability to induce filopodia. Although the head domain of Myo10 enables it to function as an actin-based motor, its tail contains binding sites for several molecules with central roles in cell biology, including phosphatidylinositol (3,4,5)-trisphosphate, microtubules and integrins. Myo10 also undergoes fascinating long-range movements within filopodia, which appear to represent a newly recognized system of transport. Myo10 is also unusual in that it is a myosin with important roles in the spindle, a microtubule-based structure. Exciting new studies have begun to reveal the structure and single-molecule properties of this intriguing myosin, as well as its mechanisms of regulation and induction of filopodia. At the cellular and organismal level, growing evidence demonstrates that Myo10 has crucial functions in numerous processes ranging from invadopodia formation to cell migration.
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Project description:Bass Myo3A, a class III myosin, was expressed in HeLa cells as a GFP fusion in order to study its cellular localization. GFP-Myo3A localized to the cytoplasm and to the tips of F-actin bundles in filopodia, a localization that is consistent with the observed concentration toward the distal ends of F-actin bundles in photoreceptor cells. A mutation in the motor active site resulted in a loss of filopodia localization, suggesting that Myo3A motor activity is required for filopodial tip localization. Deletion analyses showed that the NH2-terminal kinase domain is not required but the CO2H-terminal 22 amino acids of the Myo3A tail are required for filopodial localization. Expression of this tail fragment alone produced fluorescence associated with F-actin throughout the cytoplasm and filopodia and a recombinant tail fragment bound to F-actin in vitro. An actin-binding motif was identified within this tail fragment, and a mutation within this motif abolished both filopodia localization by Myo3A and F-actin binding by the tail fragment alone. Calmodulin localized to filopodial tips when coexpressed with Myo3A but not in the absence of Myo3A, an observation consistent with the previous proposal that class III myosins bind calmodulin and thereby localize it in certain cell types.
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Project description:The adhesion of integrins to the extracellular matrix is regulated by binding of the cytoskeletal protein talin to the cytoplasmic tail of the β-integrin subunit. Structural studies of this interaction have hitherto largely focused on the β3-integrin, one member of the large and diverse integrin family. Here, we employ NMR to probe interactions and dynamics, revealing marked structural diversity in the contacts between β1A, β1D, and β3 tails and the Talin1 and Talin2 isoforms. Coupled with analysis of recent structures of talin/β tail complexes, these studies elucidate the thermodynamic determinants of this heterogeneity and explain why the Talin2/β1D isoforms, which are co-localized in striated muscle, form an unusually tight interaction. We also show that talin/integrin affinity can be enhanced 1000-fold by deleting two residues in the β tail. Together, these studies illustrate how the integrin/talin interaction has been fine-tuned to meet varying biological requirements.
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Project description:Integrin αIIbβ3 is a predominant type of integrin abundantly expressed on the surface of platelets and its activation regulates the process of thrombosis. Talin and kindlin are cytoplasmic proteins that bind to integrin and modulate its affinity for extracellular ligands. Although the molecular details of talin-mediated integrin activation are known, the mechanism of kindlin involvement in this process remains elusive. Here, we demonstrate that the interplay between talin and kindlin promotes integrin activation. Our all-atomic molecular dynamics simulations on complete transmembrane and cytoplasmic domains of integrin αIIbβ3, talin1 F2/F3 subdomains, and the kindlin2 FERM domain in an explicit lipid-water environment over a microsecond timescale unraveled the role of kindlin as an enhancer of the talin interaction with the membrane proximal region of β-integrin. The cooperation of kindlin with talin results in a complete disruption of salt bridges between R995 on αIIb and D723/E726 on β3. Furthermore, kindlin modifies the molecular mechanisms of inside-out activation by decreasing the crossing angle between transmembrane helices of integrin αIIbβ3, which eventually results in parallelization of integrin dimer. In addition, our control simulation featuring integrin in complex with kindlin reveals that kindlin binding is not sufficient for unclasping the inner-membrane and outer-membrane interactions of integrin dimer, thus ruling out the possibility of solitary action of kindlin in integrin activation.
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Project description:Filopodia are actin-rich finger-like cytoplasmic projections extending from the leading edge of cells. Unconventional myosin-X is involved in the protrusion of filopodia. However, the underlying mechanism of myosin-X-induced filopodia formation is obscure. Here, we studied the movements of myosin-X during filopodia protrusion using a total internal reflection microscope to clarify the mechanism of myosin-X-induced filopodia formation. Myosin-X was recruited to the discrete site at the leading edge where it assembles with exponential kinetics before the filopodia extension. The myosin-X-induced filopodia showed repeated extension-retraction cycles with each extension of 2.4 microm, which was critical to produce long filopodia. Myosin-X, lacking the FERM domain, could move to the tip as does the wild type. However, it was transported toward the cell body during filopodia retraction, did not undergo multiple extension-retraction cycles, and failed to produce long filopodia. During the filopodia protrusion, the single molecules of full-length myosin-X moved within filopodia. The majority of the fluorescence spots showed two-step photobleaching, suggesting that the moving myosin-X is a dimer. Deletion of the FERM domain did not change the movement at the single molecule level with the same velocity of approximately 600 nm/s as wild-type, suggesting that the myosin-X in filopodia moves without interaction with the attached membrane via the FERM domain. Based upon these results, we have proposed a model of myosin-X-induced filopodia protrusion.
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Project description:Filopodia are actin-filled protrusions employed by cells to interact with their environment. Filopodia formation in Amoebozoa and Metazoa requires the phylogenetically diverse MyTH4-FERM (MF) myosins DdMyo7 and Myo10, respectively. While Myo10 is known to form antiparallel dimers, DdMyo7 lacks a coiled-coil domain in its proximal tail region, raising the question of how such divergent motors perform the same function. Here, it is shown that the DdMyo7 lever arm plays a role in both autoinhibition and function while the proximal tail region can mediate weak dimerization, and is proposed to be working in cooperation with the C-terminal MF domain to promote partner-mediated dimerization. Additionally, a forced dimer of the DdMyo7 motor is found to weakly rescue filopodia formation, further highlighting the importance of the C-terminal MF domain. Thus, weak dimerization activity of the DdMyo7 proximal tail allows for sensitive regulation of myosin activity to prevent inappropriate activation of filopodia formation. The results reveal that the principles of MF myosin-based filopodia formation are conserved via divergent mechanisms for dimerization.
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Project description:Cells undergo dynamic remodeling of the cytoskeleton during adhesion and migration on various extracellular matrix (ECM) substrates in response to physiological and pathological cues. The major mediators of such cellular responses are the heterodimeric adhesion receptors, the integrins. Extracellular or intracellular signals emanating from different signaling cascades cause inside-out signaling of integrins via talin, a cystokeletal protein that links integrins to the actin cytoskeleton. Various integrin subfamilies communicate with each other and growth factor receptors under diverse cellular contexts to facilitate or inhibit various integrin-mediated functions. Since talin is an essential mediator of integrin activation, much of the integrin crosstalk would therefore be influenced by talin. However, despite the existence of an extensive body of knowledge on the role of talin in integrin activation and as a stabilizer of ECM-actin linkage, information on its role in regulating inter-integrin communication is limited. This review will focus on the structure of talin, its regulation of integrin activation and discuss its potential role in integrin crosstalk. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé.
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Project description:Cell-matrix adhesion is essential for building animals, promoting tissue cohesion, and enabling cells to migrate and resist mechanical force. Talin is an intracellular protein that is critical for linking integrin extracellular-matrix receptors to the actin cytoskeleton. A key question raised by structure-function studies is whether talin, which is critical for all integrin-mediated adhesion, acts in the same way in every context. We show that distinct combinations of talin domains are required for each of three different integrin functions during Drosophila development. The partial function of some mutant talins requires vinculin, indicating that recruitment of vinculin allows talin to duplicate its own activities. The different requirements are best explained by alternative mechanisms of talin function, with talin using one or both of its integrin-binding sites. We confirmed these alternatives by showing that the proximity between the second integrin-binding site and integrins differs, suggesting that talin adopts different orientations relative to integrins. Finally, we show that vinculin and actomyosin activity help change talin's orientation. These findings demonstrate that the mechanism of talin function differs in each developmental context examined. The different arrangements of the talin molecule relative to integrins suggest that talin is able to sense different force vectors, either parallel or perpendicular to the membrane. This provides a paradigm for proteins whose apparent uniform function is in fact achieved by a variety of distinct mechanisms involving different molecular architectures.
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Project description:Cells need to sense their environment to ensure accurate targeting to specific destinations. This occurs in developing muscles, which need to attach to tendon cells before muscle contractions can begin. Elongating myotube tips form filopodia, which are presumed to have sensory roles, and are later suppressed upon building the attachment site. Here, we use live imaging and quantitative image analysis of lateral transverse (LT) myotubes in Drosophila to show that filopodia suppression occurs as a result of integrin signaling. Loss of the integrin subunits αPS2 and βPS (also known as If and Mys, respectively, in flies) increased filopodia number and length at stages when they are normally suppressed. Conversely, inducing integrin signaling, achieved by the expression of constitutively dimerised βPS cytoplasmic domain (diβ), prematurely suppressed filopodia. We discovered that the integrin signal is transmitted through the protein G protein-coupled receptor kinase interacting ArfGAP (Git) and its downstream kinase p21-activated kinase (Pak). Absence of these proteins causes profuse filopodia and prevents the filopodial inhibition mediated by diβ. Thus, integrin signaling terminates the exploratory behavior of myotubes seeking tendons, enabling the actin machinery to focus on forming a strong attachment and assembling the contractile apparatus.
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Project description:Filopodia, dynamic membrane protrusions driven by polymerization of an actin filament core, can adhere to the extracellular matrix and experience both external and cell-generated pulling forces. The role of such forces in filopodia adhesion is however insufficiently understood. Here, we study filopodia induced by overexpression of myosin X, typical for cancer cells. The lifetime of such filopodia positively correlates with the presence of myosin IIA filaments at the filopodia bases. Application of pulling forces to the filopodia tips through attached fibronectin-coated laser-trapped beads results in sustained growth of the filopodia. Pharmacological inhibition or knockdown of myosin IIA abolishes the filopodia adhesion to the beads. Formin inhibitor SMIFH2, which causes detachment of actin filaments from formin molecules, produces similar effect. Thus, centripetal force generated by myosin IIA filaments at the base of filopodium and transmitted to the tip through actin core in a formin-dependent fashion is required for filopodia adhesion.
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