Structural mechanism for bidirectional actin cross-linking by T-plastin.

To orchestrate cell mechanics, trafficking, and motility, cytoskeletal filaments must assemble into higher-order networks whose local subcellular architecture and composition specify their functions. Cross-linking proteins bridge filaments at the nanoscale to control a network's µm-scale geometry, thereby conferring its mechanical properties and functional dynamics. While these interfilament linkages are key determinants of cytoskeletal function, their structural mechanisms remain poorly understood. Plastins/fimbrins are an evolutionarily ancient family of tandem calponin-homology domain (CHD) proteins required to construct multiple classes of actin networks, which feature diverse geometries specialized to power cytokinesis, microvilli and stereocilia biogenesis, and persistent cell migration. Here, we focus on the structural basis of actin network assembly by human T-plastin, a ubiquitously expressed isoform necessary for the maintenance of stable cellular protrusions generated by actin polymerization forces. By implementing a machine-learning-enabled cryo-electron microscopy pipeline for visualizing cross-linkers bridging multiple filaments, we uncover a sequential bundling mechanism enabling T-plastin to bridge pairs of actin filaments in both parallel and antiparallel orientations. T-plastin populates distinct structural landscapes in these two bridging orientations that are selectively compatible with actin networks featuring divergent architectures and functions. Our structural, biochemical, and cell biological data highlight inter-CHD linkers as key structural elements underlying flexible but stable cross-linking that are likely to be disrupted by T-plastin mutations that cause hereditary bone diseases.

Enhanced substrate stress relaxation promotes filopodia-mediated cell migration.

Cell migration on two-dimensional substrates is typically characterized by lamellipodia at the leading edge, mature focal adhesions and spread morphologies. These observations result from adherent cell migration studies on stiff, elastic substrates, because most cells do not migrate on soft, elastic substrates. However, many biological tissues are soft and viscoelastic, exhibiting stress relaxation over time in response to a deformation. Here, we have systematically investigated the impact of substrate stress relaxation on cell migration on soft substrates. We observed that cells migrate minimally on substrates with an elastic modulus of 2 kPa that are elastic or exhibit slow stress relaxation, but migrate robustly on 2-kPa substrates that exhibit fast stress relaxation. Strikingly, migrating cells were not spread out and did not extend lamellipodial protrusions, but were instead rounded, with filopodia protrusions extending at the leading edge, and exhibited small nascent adhesions. Computational models of cell migration based on a motor-clutch framework predict the observed impact of substrate stress relaxation on cell migration and filopodia dynamics. Our findings establish substrate stress relaxation as a key requirement for robust cell migration on soft substrates and uncover a mode of two-dimensional cell migration marked by round morphologies, filopodia protrusions and weak adhesions.

T-Plastin reinforces membrane protrusions to bridge matrix gaps during cell migration.

Migrating cells move across diverse assemblies of extracellular matrix (ECM) that can be separated by micron-scale gaps. For membranes to protrude and reattach across a gap, actin filaments, which are relatively weak as single filaments, must polymerize outward from adhesion sites to push membranes towards distant sites of new adhesion. Here, using micropatterned ECMs, we identify T-Plastin, one of the most ancient actin bundling proteins, as an actin stabilizer that promotes membrane protrusions and enables bridging of ECM gaps. We show that T-Plastin widens and lengthens protrusions and is specifically enriched in active protrusions where F-actin is devoid of non-muscle myosin II activity. Together, our study uncovers critical roles of the actin bundler T-Plastin to promote protrusions and migration when adhesion is spatially-gapped.

Membrane-proximal F-actin restricts local membrane protrusions and directs cell migration.

Cell migration is driven by local membrane protrusion through directed polymerization of F-actin at the front. However, F-actin next to the plasma membrane also tethers the membrane and thus resists outgoing protrusions. Here, we developed a fluorescent reporter to monitor changes in the density of membrane-proximal F-actin (MPA) during membrane protrusion and cell migration. Unlike the total F-actin concentration, which was high in the front of migrating cells, MPA density was low in the front and high in the back. Back-to-front MPA density gradients were controlled by higher cofilin-mediated turnover of F-actin in the front. Furthermore, nascent membrane protrusions selectively extended outward from areas where MPA density was reduced. Thus, locally low MPA density directs local membrane protrusions and stabilizes cell polarization during cell migration.

EMI1 switches from being a substrate to an inhibitor of APC/CCDH1 to start the cell cycle.

Mammalian cells integrate mitogen and stress signalling before the end of G1 phase to determine whether or not they enter the cell cycle1-4. Before cells can replicate their DNA in S phase, they have to activate cyclin-dependent kinases (CDKs), induce an E2F transcription program and inactivate the anaphase-promoting complex (APC/CCDH1, also known as the cyclosome), which is an E3 ubiquitin ligase that contains the co-activator CDH1 (also known as FZR, encoded by FZR1). It was recently shown that stress can return cells to quiescence after CDK2 activation and E2F induction but not after inactivation of APC/CCDH1, which suggests that APC/CCDH1 inactivation is the point of no return for cell-cycle entry 3 . Rapid inactivation of APC/CCDH1 requires early mitotic inhibitor 1 (EMI1)3,5, but the molecular mechanism that controls this cell-cycle commitment step is unknown. Here we show using human cell models that cell-cycle commitment is mediated by an EMI1-APC/CCDH1 dual-negative feedback switch, in which EMI1 is both a substrate and an inhibitor of APC/CCDH1. The inactivation switch triggers a transition between a state with low EMI1 levels and high APC/CCDH1 activity during G1 and a state with high EMI1 levels and low APC/CCDH1 activity during S and G2. Cell-based analysis, in vitro reconstitution and modelling data show that the underlying dual-negative feedback is bistable and represents a robust irreversible switch. Our study suggests that mammalian cells commit to the cell cycle by increasing CDK2 activity and EMI1 mRNA expression to trigger a one-way APC/CCDH1 inactivation switch that is mediated by EMI1 transitioning from acting as a substrate of APC/CCDH1 to being an inhibitor of APC/CCDH1.

The function and dynamics of the apical scaffolding protein E3KARP are regulated by cell-cycle phosphorylation.

We examine the dynamics and function of the apical scaffolding protein E3KARP/NHERF2, which consists of two PDZ domains and a tail containing an ezrin-binding domain. The exchange rate of E3KARP is greatly enhanced during mitosis due to phosphorylation at Ser-303 in its tail region. Whereas E3KARP can substitute for the function of the closely related scaffolding protein EBP50/NHERF1 in the formation of interphase microvilli, E3KARP S303D cannot. Moreover, the S303D mutation enhances the in vivo dynamics of the E3KARP tail alone, whereas in vitro the interaction of E3KARP with active ezrin is unaffected by S303D, implicating another factor regulating dynamics in vivo. A-Raf is found to be required for S303 phosphorylation in mitotic cells. Regulation of the dynamics of EBP50 is known to be dependent on its tail region but modulated by PDZ domain occupancy, which is not the case for E3KARP. Of interest, in both cases, the mechanisms regulating dynamics involve the tails, which are the most diverged region of the paralogues and probably evolved independently after a gene duplication event that occurred early in vertebrate evolution.

The surprising dynamics of scaffolding proteins.

The function of scaffolding proteins is to bring together two or more proteins in a relatively stable configuration, hence their name. Numerous scaffolding proteins are found in nature, many having multiple protein-protein interaction modules. Over the past decade, examples of scaffolding complexes long thought to be stable have instead been found to be surprisingly dynamic. These studies are scattered among different biological systems, and so the concept that scaffolding complexes might not always represent stable entities and that their dynamics can be regulated has not garnered general attention. We became aware of this issue in our studies of a scaffolding protein in microvilli, which forced us to reevaluate its contribution to their structure. The purpose of this Perspective is to draw attention to this phenomenon and discuss why complexes might show regulated dynamics. We also wish to encourage more studies on the dynamics of "stable" complexes and to provide a word of caution about how functionally important dynamic associations may be missed in biochemical and proteomic studies.

Dynamics of ezrin and EBP50 in regulating microvilli on the apical aspect of epithelial cells.

Microvilli are found on the apical surface of epithelial cells. Recent studies on the microvillar proteins ezrin and EBP50 (ezrin/radixin/moesin-binding phosphoprotein of 50 kDa) have revealed both the dynamics and the regulation of microvillar components, and how a dynamic ezrin phosphocycle is necessary to confine microvilli to the apical membrane. In the present review, we first summarize the background to allow us to place these advances in context.

The tails of apical scaffolding proteins EBP50 and E3KARP regulate their localization and dynamics.

The closely related apical scaffolding proteins ERM-binding phosphoprotein of 50 kDa (EBP50) and NHE3 kinase A regulatory protein (E3KARP) both consist of two postsynaptic density 95/disks large/zona occludens-1 (PDZ) domains and a tail ending in an ezrin-binding domain. Scaffolding proteins are thought to provide stable linkages between components of multiprotein complexes, yet in several types of epithelial cells, EBP50, but not E3KARP, shows rapid exchange from microvilli compared with its binding partners. The difference in dynamics is determined by the proteins' tail regions. Exchange rates of EBP50 and E3KARP correlated strongly with their abilities to precipitate ezrin in vivo. The EBP50 tail alone is highly dynamic, but in the context of the full-length protein, the dynamics is lost when the PDZ domains are unable to bind ligand. Proteomic analysis of the effects of EBP50 dynamics on binding-partner preferences identified a novel PDZ1 binding partner, the I-BAR protein insulin receptor substrate p53 (IRSp53). Additionally, the tails promote different microvillar localizations for EBP50 and E3KARP, which localized along the full length and to the base of microvilli, respectively. Thus the tails define the localization and dynamics of these scaffolding proteins, and the high dynamics of EBP50 is regulated by the occupancy of its PDZ domains.

PDZ interactions regulate rapid turnover of the scaffolding protein EBP50 in microvilli.

Scaffolding proteins containing PDZ (postsynaptic density 95/discs large/zonula occludens-1) domains are believed to provide relatively stable linkages between components of macromolecular complexes and in some cases to bridge to the actin cytoskeleton. The microvillar scaffolding protein EBP50 (ERM-binding phosphoprotein of 50 kD), consisting of two PDZ domains and an ezrin-binding site, retains specific proteins in microvilli and is necessary for microvillar biogenesis. Our analysis of the dynamics of microvillar proteins in vivo indicated that ezrin and microvillar membrane proteins had dynamics consistent with actin treadmilling and microvillar lifetimes. However, EBP50 was highly dynamic, turning over within seconds. EBP50 turnover was reduced by mutations that inactivate its PDZ domains and was enhanced by protein kinase C phosphorylation. Using a novel in vitro photoactivation fluorescence assay, the EBP50-ezrin interaction was shown to have a slow off-rate that was dramatically enhanced in a PDZ-regulated manner by addition of cell extract to near in vivo levels. Thus, the linking of relatively stable microvillar components can be mediated by surprisingly dynamic EBP50, a finding that may have important ramifications for other scaffolding proteins.

The scaffolding protein EBP50 regulates microvillar assembly in a phosphorylation-dependent manner.

The mechanisms by which epithelial cells regulate the presence of microvilli on their apical surface are largely unknown. A potential regulator is EBP50/NHERF1 (ERM-binding phosphoprotein of 50 kD/Na(+)-H(+) exchanger regulatory factor), a microvillar scaffolding protein with two PDZ domains followed by a C-terminal ezrin-binding domain. Using RNAi and expression of RNAi-resistant EBP50 mutants we systematically show that EBP50 is necessary for microvillar assembly and requires that EBP50 has both a functional first PDZ domain and an ezrin-binding site. Expression of mutants mimicking Cdc2 or PKC phosphorylation are nonfunctional in microvillar assembly. Biochemical analysis reveals that these mutants are defective in PDZ1 accessibility when PDZ2 is occupied, and can be rendered functional in vivo by additional mutation of PDZ2. EBP50 is not necessary for mitotic cell microvilli, and PKC activation causes a rearrangement of microvilli on cells due to phosphorylation-dependent loss of EBP50 function. Thus, EBP50 is a critical factor that regulates microvilli assembly and whose activity is regulated by signaling pathways and occupation of its PDZ2 domain.

A regulated complex of the scaffolding proteins PDZK1 and EBP50 with ezrin contribute to microvillar organization.

PDZK1 and ezrin, radixin, moesin binding phosphoprotein 50 kDa (EBP50) are postsynaptic density 95/disc-large/zona occludens (PDZ)-domain-containing scaffolding proteins found in the apical microvilli of polarized epithelial cells. Binary interactions have been shown between the tail of PDZK1 and the PDZ domains of EBP50, as well as between EBP50 and the membrane-cytoskeletal linking protein ezrin. Here, we show that these molecules form a regulated ternary complex in vitro and in vivo. Complex formation is cooperative because ezrin positively influences the PDZK1/EBP50 interaction. Moreover, the interaction of PDZK1 with EBP50 is enhanced by the occupancy of EBP50's adjacent PDZ domain. The complex is further regulated by location, because PDZK1 shuttles from the nucleus in low confluence cells to microvilli in high confluence cells, and this regulates the formation of the PDZK1/EBP50/ezrin complex in vivo. Knockdown of EBP50 decreases the presence of microvilli, a phenotype that can be rescued by EBP50 re-expression or expression of a PDZK1 chimera that is directly targeted to ezrin. Thus, when appropriately located, PDZK1 can provide a function necessary for microvilli formation normally provided by EBP50. By entering into the ternary complex, PDZK1 can both enhance the scaffolding at the apical membrane as well as augment EBP50's role in microvilli formation.

EPI64 regulates microvillar subdomains and structure.

EPI64 is a TBC domain-containing protein that binds the PDZ domains of EBP50, which binds ezrin, a major actin-binding protein of microvilli. High-resolution light microscopy revealed that ezrin and EBP50 localize exclusively to the membrane-surrounded region of microvilli, whereas EPI64 localizes to variable regions in the structures. Overexpressing EPI64 results in its and EBP50's relocalization to the base of microvilli, including to the actin rootlet devoid of ezrin or plasma membrane. Uncoupling EPI64's binding to EBP50, expression of any construct mislocalizing its TBC domain, or knock down of EBP50 results in loss of microvilli. The TBC domain of EPI64 binds directly to Arf6-GTP. Overexpressing the TBC domain increases Arf6-GTP levels, and expressing dominant-active Arf6 results in microvillar loss. These data reveal that microvilli have distinct cytoskeletal subdomains and that EPI64 regulates microvillar structure.

Epidermal growth factor receptor downregulation in cultured bovine cumulus cells: reconstitution of calcium signaling and stimulated membrane permeabilization.

Cumulus cell-oocyte complexes (COCs), cultured in vitro, are competent for maturation and fertilization. Inclusion of epidermal growth factor (EGF) in the COC culture medium enhances in vitro maturation and subsequent embryonic development. It has been shown that isolated COCs exposed to EGF respond with a prolonged and pulsatile release of Ca2+ into the extra-cellular medium and that cumulus cells (CCs) of complexes exhibit both a slow rise in intracellular [Ca2+] ([Ca2+]i) and plasma membrane permeabilization in response to EGF. These unusual signaling responses were examined in isolated, cultured bovine CCs. Few individual CCs showed [Ca2+]i increases; the lack of response was found to be due to decrease of expression of endogenous EGF receptors after dissociation. CCs transfected with a human EGF receptor-GFP fusion protein showed robust, prolonged, EGF-stimulated [Ca2+]i elevations characteristic of CC responses in intact COCs. Many CCs that responded to EGF stimulation with a [Ca2+]i rise also released entrapped fura-2 dye at the peak of the [Ca2+]i response, suggesting that CC permeabilization and death follows activation of the EGF receptor. The [Ca2+]i elevation due to EGF stimulation and subsequent membrane permeabilization was shown to be mediated by the inositol triphosphate signaling pathway.