Parallel Actin Bundles Research Articles

Myo10 tail is crucial for promoting long filopodia

Filopodia are slender cellular protrusions containing parallel actin bundles involved in environmental sensing and signaling, cell adhesion and migration, and growth cone guidance and extension. Myosin 10 (Myo10), an unconventional actin-based motor protein, was reported to induce filopodial initiation with its motor domain. However, the roles of the multifunctional tail domain of Myo10 in filopodial formation and elongation remain elusive. Herein, we generated several constructs of Myo10-full-length Myo10, Myo10 with a truncated tail (Myo10 HMM), and Myo10 containing four mutations to disrupt its coiled-coil domain (Myo10 CC mutant). We found that the truncation of the tail domain decreased filopodial formation and filopodial length, while four mutations in the coiled-coil domain disrupted the motion of Myo10 toward filopodial tips and the elongation of filopodia. Furthermore, we found that filopodia elongated through multiple elongation cycles, which was supported by the Myo10 tail. These findings suggest that Myo10 tail is crucial for promoting long filopodia.

Myosin V facilitates polarised E-cadherin secretion.

E-cadherin has a fundamental role in epithelial tissues by providing cell-cell adhesion. Polarised E-cadherin exocytosis to the lateral plasma membrane is central for cell polarity and epithelial homeostasis. Loss of E-cadherin secretion compromises tissue integrity and is a prerequisite for metastasis. Despite this pivotal role of E-cadherin secretion, the transport mechanism is still unknown. Here we identify Myosin V as the motor for E-cadherin secretion. Our data reveal that Myosin V and F-actin are required for the formation of a continuous apicolateral E-cadherin belt, the zonula adherens. We show by live imaging how Myosin V transports E-cadherin vesicles to the plasma membrane, and distinguish two distinct transport tracks: an apical actin network leading to the zonula adherens and parallel actin bundles leading to the basal-most region of the lateral membrane. E-cadherin secretion starts in endosomes, where Rab11 and Sec15 recruit Myosin V for transport to the zonula adherens. We also shed light on the endosomal sorting of E-cadherin by showing how Rab7 and Snx16 cooperate in moving E-cadherin into the Rab11 compartment. Thus, our data help to understand how polarised E-cadherin secretion maintains epithelial architecture and prevents metastasis.

Nonmuscle myosin-2 contractility-dependent actin turnover limits the length of epithelial microvilli.

Brush border microvilli enable functions that are critical for epithelial homeostasis, including solute uptake and host defense. However, the mechanisms that regulate the assembly and morphology of these protrusions are poorly understood. The parallel actin bundles that support microvilli have their pointed-end rootlets anchored in a filamentous meshwork referred to as the “terminal web.” Although classic electron microscopy studies revealed complex ultrastructure, the composition and function of the terminal web remain unclear. Here we identify nonmuscle myosin-2C (NM2C) as a component of the terminal web. NM2C is found in a dense, isotropic layer of puncta across the subapical domain, which transects the rootlets of microvillar actin bundles. Puncta are separated by ∼210 nm, the expected size of filaments formed by NM2C. In intestinal organoid cultures, the terminal web NM2C network is highly dynamic and exhibits continuous remodeling. Using pharmacological and genetic perturbations in cultured intestinal epithelial cells, we found that NM2C controls the length of growing microvilli by regulating actin turnover in a manner that requires a fully active motor domain. Our findings answer a decades-old question on the function of terminal web myosin and hold broad implications for understanding apical morphogenesis in diverse epithelial systems.

Upregulation of fascin-1 is involved in HIF-1α-dependent invasion and migration of hypopharyngeal squamous cell carcinoma.

Fascin-1 is an actin-bundling protein, which specifically interacts with F-actin to form parallel actin bundles, and participates in the regulation of cell adhesion, interactions and migration. However, the expression and regulatory mechanisms of fascin-1 in hypopharyngeal squamous cell carcinoma (HSCC) remain poorly understood. The present study investigated the effects and underlying molecular mechanism of fascin-1 on the invasion and metastasis of HSCC. The results demonstrated that fascin-1 was overexpressed and correlated with lymph node metastasis and tumor-node-metastasis stage in HSCC tissues. Further in vitro study revealed that fascin-1 promoted cell morphology polarization to increase the motility of FaDu cells. In addition, fascin-1 significantly promoted the migration and invasion of FaDu cells. At the molecular level, fascin-1 promoted cell invasion and migration by upregulating matrix metalloproteinase-2 (MMP-2) expression in FaDu cells. Immunohistochemical analysis revealed that a correlation existed between hypoxia inducible factor (HIF)-1α and fascin-1 expression in the HSCC tissues. Furthermore, the results from a cobalt chloride-induced hypoxia model demonstrated that fascin-1 may be upregulated by HIF-1α in FaDu cells. Further analysis revealed that fascin-1 knockdown significantly decreased the invasion of cells under hypoxia and partially reversed hypoxia-induced MMP-2 expression under hypoxia in FaDu cells. In conclusion, fascin-1 was upregulated by HIF-1α, and promoted the invasion and migration of HSCC cells; therefore, fascin-1 may provide a potential target for the treatment of invasion and metastasis in HSCC.

Correlative cryo-electron microscopy reveals the structure of TNTs in neuronal cells

The orchestration of intercellular communication is essential for multicellular organisms. One mechanism by which cells communicate is through long, actin-rich membranous protrusions called tunneling nanotubes (TNTs), which allow the intercellular transport of various cargoes, between the cytoplasm of distant cells in vitro and in vivo. With most studies failing to establish their structural identity and examine whether they are truly open-ended organelles, there is a need to study the anatomy of TNTs at the nanometer resolution. Here, we use correlative FIB-SEM, light- and cryo-electron microscopy approaches to elucidate the structural organization of neuronal TNTs. Our data indicate that they are composed of a bundle of open-ended individual tunneling nanotubes (iTNTs) that are held together by threads labeled with anti-N-Cadherin antibodies. iTNTs are filled with parallel actin bundles on which different membrane-bound compartments and mitochondria appear to transfer. These results provide evidence that neuronal TNTs have distinct structural features compared to other cell protrusions.

Human papilloma virus infection and fascin over-expression in squamous cell carcinoma of the cervix.

Background: Human papilloma virus (HPV) is involved in development of almost all cervical cancers, mainly through the subversion of cellular mechanisms of growth control. Fascin plays central role in subsequent cell transformation events. Fascin mediates stabilization of parallel actin bundles where cellular protrusions are formed; this represents primary stages of cell migration and metastasis. Immunohistochemical assays have shown up-regulation of fascin expression in many epithelial and non-epithelial neoplasms. Therefore, the aim of this study was to investigate HPV infection and fascin expression in samples of cervical cancer. Methods: Of 66 patients with confirmed SCC, formalin-fixed specimens, embedded in paraffin blocks were evaluated for HPV infection with nested multiplex polymerase chain reaction (NM-PCR) and for fascin expression with immunohistochemical assays. Statistical analysis was performed using Wilcoxon rank-sum test and SPSS software. A p<0.05 was considered for statistical significance. Results: Of 66 samples, 52 (78.7%) were found positive for HPV infection and fascin over-expression was shown in all squamous cell carcinoma samples. Conclusion: This study showed fascin overexpression in squamous cell carcinoma of the cervix which might be involved in metastasis of cancers induced by some types of HPV, hypothetically through attenuation of inter-cellular adhesions, and induction of cell motility

Palladin Compensates for the Arp2/3 Complex and Supports Actin Structures during Listeria Infections.

ABSTRACTPalladin is an important component of motile actin-rich structures and nucleates branched actin filament arrays in vitro. Here we examine the role of palladin during Listeria monocytogenes infections in order to tease out novel functions of palladin. We show that palladin is co-opted by L. monocytogenes during its cellular entry and intracellular motility. Depletion of palladin resulted in shorter and misshapen comet tails, and when actin- or VASP-binding mutants of palladin were overexpressed in cells, comet tails disintegrated or became thinner. Comet tail thinning resulted in parallel actin bundles within the structures. To determine whether palladin could compensate for the Arp2/3 complex, we overexpressed palladin in cells treated with the Arp2/3 inhibitor CK-666. In treated cells, bacterial motility could be initiated and maintained when levels of palladin were increased. To confirm these findings, we utilized a cell line depleted of multiple Arp2/3 complex subunits. Within these cells, L. monocytogenes failed to generate comet tails. When palladin was overexpressed in this Arp2/3 functionally null cell line, the ability of L. monocytogenes to generate comet tails was restored. Using purified protein components, we demonstrate that L. monocytogenes actin clouds and comet tails can be generated (in a cell-free system) by palladin in the absence of the Arp2/3 complex. Collectively, our results demonstrate that palladin can functionally replace the Arp2/3 complex during bacterial actin-based motility.

Filopodia-Like Structure Formation from Xenopus Egg Extracts.

The actin cytoskeleton comprises many different architectures of filaments, including branched networks, parallel bundles and antiparallel fibers. A current challenge is to elucidate how the diverse array of actin regulators, which controls the growth, assembly and turnover of actin filaments, is used to orchestrate cytoskeletal organization and in turn cell shape and movement. Long observed to assemble at cell membranes, actin in Xenopus egg extracts recapitulates membrane-triggered assembly at specific lipid and membrane environments. The use of Xenopus egg extracts has contributed greatly to identifying how constitutively autoinhibited regulatory pathways are activated, which converge on activation of the Arp2/3 complex. Here we describe a protocol for making parallel actin bundles using Xenopus egg extracts from supernatants prepared by high-speed centrifugation. These filopodia-like actin bundles emanate from clusters of actin regulators that self-assemble at phosphatidylinositol (4,5)-bisphosphate-containing supported lipid bilayers. Forming a plasma membrane-mimicking bilayer on glass allows easy, optimizable, high signal-to-noise microscopy at high spatial and temporal resolution. The use of Xenopus egg extracts yields large quantities of active material that can be flexibly tailored to address specific questions, for example, by dilution, addition of fluorescent proteins, antibodies or protein fragments, immunodepletion, addition of small molecule inhibitors, or biochemical fractionation.

Long-range self-organization of cytoskeletal myosin II filament stacks.

Although myosin II filaments are known to exist in non-muscle cells, their dynamics and organization are incompletely understood. Here, we combined structured illumination microscopy with pharmacological and genetic perturbations, to study the process of actomyosin cytoskeleton self-organization into arcs and stress fibres. A striking feature of the myosin II filament organization was their 'registered' alignment into stacks, spanning up to several micrometres in the direction orthogonal to the parallel actin bundles. While turnover of individual myosin II filaments was fast (characteristic half-life time 60 s) and independent of actin filament turnover, the process of stack formation lasted a longer time (in the range of several minutes) and required myosin II contractility, as well as actin filament assembly/disassembly and crosslinking (dependent on formin Fmnl3, cofilin1 and α-actinin-4). Furthermore, myosin filament stack formation involved long-range movements of individual myosin filaments towards each other suggesting the existence of attractive forces between myosin II filaments. These forces, possibly transmitted via mechanical deformations of the intervening actin filament network, may in turn remodel the actomyosin cytoskeleton and drive its self-organization.

Force Matters: Biomechanical Regulation of Cell Invasion and Migration in Disease.

Atherosclerosis, cancer, and various chronic fibrotic conditions are characterized by an increase in the migratory behavior of resident cells and the enhanced invasion of assorted exogenous cells across a stiffened extracellular matrix (ECM). This stiffened scaffold aberrantly engages cellular mechanosignaling networks in cells, which promotes the assembly of invadosomes and lamellae for cell invasion and migration. Accordingly, deciphering the conserved molecular mechanisms whereby matrix stiffness fosters invadosome and lamella formation could identify therapeutic targets to treat fibrotic conditions, and reducing ECM stiffness could ameliorate disease progression.

Comparing the Motility of Myosin X with Parallel and Anti-Parallel Dimerization Domains

Myosin X is an actin-based molecular motor involved in filopodial formation, cell migration, adhesion and mitotic spindle orientation. It is upregulated in some cancers and may play a role in metastasis. Myosin X induces filopodial formation and localizes to filopodial tips by an unclear targeting mechanism. Studies using various truncated, forced-dimers have indicated that processivity of myosin X is greater on fascin-bundled actin filaments than single filaments, whereas other studies have not found different processivity on bundles relative to single filaments. Recently, the native dimerization domain has been proposed to form anti-parallel dimers, which might influence aspects of motility such as preference for actin bundles. To test this idea, we compared processive motility of forced parallel and anti-parallel myosin X constructs on single and bundled actin filaments. On single actin filaments parallel constructs are robustly processive, while processivity of the anti-parallel construct is much weaker. Run lengths for anti-parallel myosin X decreased as [MgATP] was raised from 0.5 to 2.5 micromolar. At 5 micromolar MgATP, run lengths are shorter than our detection limit, ∼100 nm. The velocity of the anti-parallel construct on single actin filaments is slower than that of the parallel construct, due to a substantial proportion of short (e.g. +18 nm) and backward steps along with +36 nm steps. On fascin-actin bundles, however, the processivity, velocity, step-size, and backward stepping frequency are similar to those characteristics of parallel myosin X. These results suggest that in cells, the anti-parallel coiled-coil domain may help myosin X select structures containing parallel actin bundles, such as filopodia. Supported by NIH grant R01GM086352 and T32HL07954 and TEXAS STAR Plus award.

Characterization of a Unique Myosin IIIa Deafness Mutation which Enhances Actin-Sliding Velocity but Abolishes Filopodia Tip Localization

MYO3A, an unconventional myosin that localizes to the distal tips of stereocilia in the sensory epithelia of the vertebrate auditory and vestibular systems, consists of an N-terminal kinase domain, an actin-activated motor domain, and a class-specific tail domain. Previous work indicates that MYO3A tip-localization is regulated through autophosphorylation. MYO3A is thought to transport the actin regulatory protein, ESPIN1, to the stereocilia tips in inner ear hair cells and mutations in MYO3A lead to non-syndromic deafness in humans. We investigated the G488E mutation in MYO3A, a deafness associated mutation located near the conserved switch I region. The G488E mutation reduces the maximum actin-activated ATPase rate 2-fold and increases 10-fold the actin concentration required to reach one-half maximal ATPase. Interestingly, G488E MYO3A increases 2-fold the actin filament sliding velocity measured with the in vitro motility assay. We also investigated the impact of the G488E mutation on cellular localization by examining GFP tagged MYO3A dK constructs transfected into COS7 cells. The G488E mutation completely abolished MYO3A dK filopodia tip localization both in the presence and absence of ESPIN1. In addition, co-transfection of G488E MYO3A dK, ESPIN1 and wild-type (WT) MYO3A dK demonstrated that the presence of WT MYO3A does not rescue the tip localization of G488E. We hypothesize that the G488E mutation disrupts the duty ratio which prevents processive movement to the tips of parallel actin bundles. In support, chimeras of the MYO3A tail with other myosin motors (MYO1A, MYO19, MYO5A, and MYO10) demonstrate that only high duty-ratio motors are capable of filopodia tip localization. Our results demonstrate that intra-filopodia motility requires high duty ratio motor activity, which may be crucial for the formation, maintenance, and ultrastructure of parallel actin bundles.

The cuticular plate: a riddle, wrapped in a mystery, inside a hair cell.

The mechanosensitive hair cells of the inner ear are crucial to hearing and vestibular function. Each hair cell detects the mechanical stimuli associated with sound or head movement with a hair bundle at the apical surface of the cell, consisting of a precise array of actin-based stereocilia. Each stereocilium inserts as a rootlet into a dense filamentous actin mesh known as the cuticular plate. Disruption of the parallel actin bundles forming the stereocilia results in hearing impairments and balance defects. The cuticular plate is thought to be involved in holding the stereocilia in place. However, the precise role of the cuticular plate in hair bundle development, maintenance, and hearing remains unknown. Ultrastructural studies have revealed a complex cytoskeletal architecture, but a lack of knowledge of proteins that inhabit the cuticular plate and a dearth of mutations that perturb relevant proteins have hindered our understanding of the functions of the cuticular plate. Here, we discuss what is known about the structure and development of this unique and poorly-understood actin-rich organelle.

A role for fascin in preventing filopodia breakage in Drosophila tracheal cells

Filopodia are long and thin finger-like protrusions essential for cell migration. They are formed by parallel actin bundles tightly packed by cell type and context dependent actin-bundling proteins. Our recent work analyzing the role of Fascin during tracheal development in Drosophila has shown that Singed (the Drosophila Fascin homolog) acts as a molecular link between the Branchless (FGF)/Breathless (FGFR) pathway and the actin cytoskeleton. We have reported that the lack of Singed (Sn) leads to wavy and flaccid filopodia due to the disorganization of the tracheal actin cytoskeleton. Here we describe for the first time filopodia breakage in Drosophila, and show that Fascin plays a role in this event. We propose that actin filaments in sn mutant filopodia buckle under membrane pressure due to lower bending stiffness, eventually undergoing breakage. Both Filopodia buckling and breakage would impair correct cell navigation and migration.

Fascin, may the Forked be with you

The FGFR pathway triggers a wide range of key biological responses. Among others, the Breathless (Btl, Drosophila FGFR1) receptor cascade promotes cell migration during embryonic tracheal system development. However, how the actin cytoskeleton responds to Btl pathway activation to induce cell migration has remained largely unclear. Our recent results shed light into this issue by unveiling a link between the actin-bundling protein Singed (Sn) and the Btl pathway. We showed that the Btl pathway regulates sn, which leads to the stabilization of the actin bundles required for filopodia formation and actin cytoskeleton rearrangement. This regulation contributes to tracheal migration, tracheal branch fusion and tracheal cell elongation. Parallel actin bundles (PABs) are usually cross-linked by more than one actin-bundling protein. Accordingly, we have also shown that sn synergistically interacts with forked (f), another actin crosslinker. In this Extra View we extend f analysis and hypothesize how both actin-bundling proteins may act together to regulate the PABs during tracheal embryonic development. Although both proteins are required for similar tracheal events, we suggest that Sn is essential for actin bundle initiation and stiffening, while F is required for the lengthening and further stabilization of the PABs.

Dynamics of brush border remodeling induced by enteropathogenic E. coli

Enteropathogenic Escherichia coli (EPEC) induces dramatic remodeling of enterocyte brush borders, a process that includes microvillar effacement and actin pedestal formation. Although the Arp2/3 complex is involved in formation of a branched actin network within pedestals, the fate of parallel actin bundles in microvilli during infection remains unclear. Here, we find that in polarized intestinal epithelial cells, EPEC stimulates long-range microvillar dynamics, pulling protrusions toward sites of bacterial attachment in a process mediated by the adhesion molecule protocadherin-24. Additionally, retraction of the EPEC bundle forming pilus stimulates directed elongation of nearby microvilli. These processes lead to coalescence of microvilli and incorporation of the underlying parallel actin bundles into pedestals. Furthermore, stabilization of microvillar actin bundles delays pedestal formation. Together, these results suggest a model where EPEC takes advantage of pre-existing actin filaments in microvillar core bundles to facilitate pedestal formation.

Staurosporine‐induced collapse of cochlear hair bundles

Early postnatal mouse cochlear cultures were treated with a small panel of kinase inhibitors to elucidate the mechanisms underlying the maintenance of hair-bundle structure in the developing inner ear. At low concentrations (1–10 nM), staurosporine causes the collapse and loss of hair bundles without provoking hair-cell death, as judged by lack of terminal transferase dUTP nick end labeling (TUNEL) labeling or reactivity to anti-activated caspase-3. Staurosporine exposure results in the fusion of the hair bundle’s stereocilia, a resorption of the parallel actin bundles of the stereocilia into the cytoplasm of the hair cell, a detachment of the apical, non-stereociliary membrane of the hair cell from the underlying cuticular plate, and a severing of the hair-bundle’s rootlets from the actin cores of the stereocilia. It does not block membrane retrieval at the apical pole of the hair cells, nor does it elicit the externalization of phosphatidylserine. Staurosporine treatment causes a reduction in levels of the phosphorylated forms of ezrin, radixin, and moesin in cochlear cultures during the period of hair-bundle loss, indicating the integrity of the hair bundle may be actively maintained by the phosphorylation status of these proteins. J. Comp. Neurol. 522:3281–3294, 2014. © 2014 Wiley Periodicals, Inc.

Myth-Ferm Myosins have Roles in Regulating Actin Polymerization

The MyTH-FERM (MF) myosins are a subgroup of actin-based motors characterized by the presence of one or two adjacent MyTH (myosin tail homology 4)/FERM (band 4.1, ezrin, radixin, moesin) domains in their C-terminal tail regions. Although the MF myosins Myo7, Myo10, Myo15 and Myo44 are phylogenetically distinct, they have several characteristics in common. Each of these MF myosins has a striking localization to the tips of structures formed by parallel actin bundles such as filopodia and stereocilia and the different myosins play a role in the extension of either filopodia (Myo10) or stereocilia (Myo7A, Myo15) in Metazoa and filopod extension in Amoebozoa (Myo7). Their shared roles in the extension of parallel actin bundles is consistent with each of these myosins transporting or anchoring regulators of actin polymerization to the growing tip. Surprisingly, the amoeboid Myo44 is not needed for the formation of filopodia. However, Myo44 is rapidly recruited to the plasma membrane following chemotactic stimulation, consistent with its role in mediating the transmission of chemotactic signals that generate cell polarity to the actin cytoskeleton. Myo44 is necessary for the rapid activation of actin polymerization (∼5 sec) following chemotactic stimulation and is required for activating Ras, a critical small GTPase that promotes the rapid, localized polymerization of actin at side of the cell closest to the stimulus. These results establish a new role for MF myosins in signaling to the actin cytoskeleton and establish a general role for this group of myosins in regulating actin polymerization.This work was supported by grants from the NSF and AHA.

Directed Actin Assembly and Contractility

The organization of actin filaments into higher-ordered structures governs eukaryotic cell shape and movement. Global actin network size and architecture is maintained in a dynamic steady-state through regulated assembly and disassembly. We have developed a micropatterning method that enables the spatial control of actin nucleation sites for in vitro assays (Reymann, Nat Mat, 2010). These actin templates were used to evaluate the response of oriented actin structures to myosin-induced contractility. We determine that myosins selectively contract and disassemble anti-parallel actin structures while parallel actin bundles remain unaffected. In addition, the local distribution of nucleation sites and the resulting orientation of actin filaments regulate the scalability of the contraction process. This “orientation selection” mechanism for selective contraction and disassembly reveals how the dynamics of the cellular actin cytoskeleton is spatially controlled by actomyosin contractility. Further application of the micropatterning method will be presented in particular recent data on the reconstitution of a lamellipodium-type of actin organization and the fabrication of three-dimensional electrical connections by means of directed actin self-organization.

Myosin X dimerization and its impact on cellular functions

All members of the myosin superfamily of actin-based molecular motors are thought to use the energy from ATP hydrolysis to generate force and motion. Some myosins are capable of forming parallel coiled-coil dimers by virtue of a coiled-coil domain that is C-terminal to the motor catalytic region (head) (1). This form of dimerization is thought to be essential for the hand-over-hand processive walking mechanism of myosins that function as intracellular transporters (2). In this walking mechanism the mechanochemical properties of the motors are such that the likelihood of at least one head of the dimer being attached to the actin filament is high, owing to a step size that closely matches the helical pitch of the actin filament and a strain-gating mechanism in which the enzymatic cycles of the two heads are coordinated (2). Processive walking allows for multiple successive steps and long-distance transport along a single actin filament. In a study presented in PNAS, Lu et al. (3) demonstrate an antiparallel dimerization mechanism in myosin X (MYO10) and suggest that this structural arrangement allows more efficient processive walking in the parallel actin bundles of filopodia. In addition, Lu et al. (3) demonstrate that formation of the antiparallel coiled coil enhances the ability of MYO10 to induce filopodia formation.