FCHSD2 cooperates with CDC42 and N-WASP to regulate cell protrusion formation

Actin-based, finger-like cell protrusions such as microvilli and filopodia play important roles in epithelial cells. Several proteins have been identified to regulate cell protrusion formation, which helps us to learn about the underlying mechanism of this process. FCH domain and double SH3 domains containing protein 2 (FCHSD2) belongs to the FCH and Bin-Amphiphysin-Rvs (F-BAR) protein family, containing an N-terminal F-BAR domain, two SH3 domains, and a C-terminal PDZ domain-binding interface (PBI). Previously, we found that FCHSD2 interacts with WASP/N-WASP and stimulates ARP2/3-mediated actin polymerization in vitro. In the present work, we show that FCHSD2 promotes the formation of apical and lateral cell protrusions in cultured cells. Our data suggest that FCHSD2 cooperates with CDC42 and N-WASP in regulating apical cell protrusion formation. In line with this, biochemical studies reveal that FCHSD2 and CDC42 simultaneously bind to N-WASP, forming a protein complex. Interestingly, the F-BAR domain of FCHSD2 induces lateral cell protrusion formation independently of N-WASP. Furthermore, we show that the ability of FCHSD2 to induce cell protrusion formation requires its plasma membrane-binding ability. In summary, our present work suggests that FCHSD2 cooperates with CDC42 and N-WASP to regulate cell protrusion formation in a membrane-dependent manner.     

Biochemical and functional significance of F-BAR domain proteins interaction with WASP/N-WASP

The Bin-Amphiphysin-Rvs (BAR) domain family of proteins includes groups which promote positive (classical BAR, N-BAR, and F-BAR) and negative (I-BAR) membrane deformation. Of these groups, the F-BAR subfamily is the most diverse in its biochemical properties. F-BAR domain proteins dimerize to form a tight scaffold about the membrane. The F-BAR domain provides a banana-shaped, alpha-helical structure that senses membrane curvature. Different types of F-BAR domain proteins contain tyrosine kinase or GTPase activities; some interact with phosphatases and RhoGTPases. Most possess an SH3 domain that facilitates the recruitment and activation of WASP/N-WASP. Thus, F-BAR domain proteins affect remodeling of both membrane and the actin cytoskeleton. The purpose of this review is to highlight the role of F-BAR proteins in coupling WASP/N-WASP to cytoskeletal remodeling. A role for F-BAR/WASP interaction in human diseases affecting nervous, blood, and neoplastic tissues is discussed.     

SNX9 couples actin assembly to phosphoinositide signals and is required for membrane remodeling during endocytosis

Multiple modes of endocytosis require actin-dependent remodeling of the plasma membrane; however, neither the factors linking these processes nor their mechanisms of action are understood. The sorting nexin, SNX9, localizes to clathrin-coated pits where it interacts with dynamin and functions in clathrin-mediated endocytosis. Here, we demonstrate that SNX9 also localizes to actin-rich structures implicated in fluid-phase uptake, including tubular membranes containing GPI-anchored proteins and dorsal membrane ruffles. Moreover, we show that SNX9 is critical for dorsal ruffle formation and for clathrin-independent, actin-dependent fluid-phase endocytosis. In vitro, SNX9 directly associates with N-WASP, an Arp2/3 complex activator, and stimulates N-WASP/Arp2/3-mediated actin assembly. SNX9-stimulated actin polymerization is greatly enhanced by PI(4,5)P(2)-containing liposomes, due in part to PI(4,5)P(2)-induced SNX9 oligomerization. These results suggest a mechanism for the spatial and temporal regulation of N-WASP-dependent actin assembly and implicate SNX9 in directly coupling actin dynamics to membrane remodeling during multiple modes of endocytosis.     

ANKRD24 organizes TRIOBP to reinforce stereocilia insertion points

The stereocilia rootlet is a key structure in vertebrate hair cells, anchoring stereocilia firmly into the cell’s cuticular plate and protecting them from overstimulation. Using superresolution microscopy, we show that the ankyrin-repeat protein ANKRD24 concentrates at the stereocilia insertion point, forming a ring at the junction between the lower and upper rootlets. Annular ANKRD24 continues into the lower rootlet, where it surrounds and binds TRIOBP-5, which itself bundles rootlet F-actin. TRIOBP-5 is mislocalized in Ankrd24^KO/KO hair cells, and ANKRD24 no longer localizes with rootlets in mice lacking TRIOBP-5; exogenous DsRed–TRIOBP-5 restores endogenous ANKRD24 to rootlets in these mice. Ankrd24^KO/KO mice show progressive hearing loss and diminished recovery of auditory function after noise damage, as well as increased susceptibility to overstimulation of the hair bundle. We propose that ANKRD24 bridges the apical plasma membrane with the lower rootlet, maintaining a normal distribution of TRIOBP-5. Together with TRIOBP-5, ANKRD24 organizes rootlets to enable hearing with long-term resilience.     

SNX9 activities are regulated by multiple phosphoinositides through both PX and BAR domains

Sorting nexin 9 (SNX9) functions at the interface between membrane remodeling and the actin cytoskeleton. In particular, SNX9 links membrane binding to potentiation of N-WASP and dynamin GTPase activities. SNX9 is one of a growing number of proteins that contain two lipid-binding domains, a phox homology (PX) and a Bin1/Amphiphysin/RVS167 (BAR) domain, and localizes to diverse membranes that are enriched in different phosphoinositides. Here, we investigate the mechanism by which SNX9 functions at these varied membrane environments. We show that SNX9 has low-lipid-binding affinity and harnesses a broad range of phosphoinositides to synergistically enhance both dynamin and N-WASP activities. We introduced point mutations in either the PX domain, BAR domain or both that are predicted to disrupt their functions and examined their respective roles in lipid-binding, and dynamin and N-WASP activation. We show that the broad lipid specificity of SNX9 is not because of independent and additive contributions by individual domains. Rather, the two domains appear to function in concert to confer lipid-binding and SNX9's membrane active properties. We also demonstrate that the two domains are differentially required for full SNX9 activity in N-WASP and dynamin regulation, and for localization of SNX9 to clathrin-coated pits and dorsal ruffles. In total, our results suggest that SNX9 can integrate signals from varied lipids through two domains to direct membrane remodeling events at multiple cellular locations.     

Erk/Src phosphorylation of cortactin acts as a switch on-switch off mechanism that controls its ability to activate N-WASP

The Arp2/3 complex can be independently activated to initiate actin polymerization by the VCA domain of WASP family members and by the acidic N-terminal and F-actin-binding repeat region of cortactin, which possesses a C-terminal SH3 domain. Cortactin is a target for phosphorylation by Src tyrosine kinases and by serine/threonine kinases that include Erk. Here we demonstrate that cortactin binds N-WASP and WASP via its SH3 domain, induces in vitro N-WASP-mediated actin polymerization, and colocalizes with N-WASP and WASP at sites of active actin polymerization. Erk phosphorylation and a mimicking S405,418D double mutation enhanced cortactin binding and activation of N-WASP. In contrast, Src phosphorylation inhibited the ability of cortactin previously phosphorylated by Erk, and that of S405,418D double mutant cortactin, to bind and activate N-WASP. Furthermore, Y-->D mutation of three tyrosine residues targeted by Src (Y421, Y466, and Y482) inhibited the ability of S405,418D cortactin to activate N-WASP. We propose that Erk phosphorylation liberates the SH3 domain of cortactin from intramolecular interactions with proline-rich regions, causing it to synergize with WASP and N-WASP in activating the Arp2/3 complex, and that Src phosphorylation terminates cortactin activation of N-WASP and WASP.     

The type III effector EspF coordinates membrane trafficking by the spatiotemporal activation of two eukaryotic signaling pathways

Bacterial toxins and effector proteins hijack eukaryotic enzymes that are spatially localized and display rapid signaling kinetics. However, the molecular mechanisms by which virulence factors engage highly dynamic substrates in the host cell environment are poorly understood. Here, we demonstrate that the enteropathogenic Escherichia coli (EPEC) type III effector protein EspF nucleates a multiprotein signaling complex composed of eukaryotic sorting nexin 9 (SNX9) and neuronal Wiskott-Aldrich syndrome protein (N-WASP). We demonstrate that a specific and high affinity association between EspF and SNX9 induces membrane remodeling in host cells. These membrane-remodeling events are directly coupled to N-WASP/Arp2/3-mediated actin nucleation. In addition to providing a biochemical mechanism of EspF function, we find that EspF dynamically localizes to membrane-trafficking organelles in a spatiotemporal pattern that correlates with SNX9 and N-WASP activity in living cells. Thus, our findings suggest that the EspF-dependent assembly of SNX9 and N-WASP represents a novel form of signaling mimicry used to promote EPEC pathogenesis and gastrointestinal disease.     

The F-BAR Cdc15 promotes contractile ring formation through the direct recruitment of the formin Cdc12

In Schizosaccharomyces pombe, cytokinesis requires the assembly and constriction of an actomyosin-based contractile ring (CR). Nucleation of F-actin for the CR requires a single formin, Cdc12, that localizes to the cell middle at mitotic onset. Although genetic requirements for formin Cdc12 recruitment have been determined, the molecular mechanisms dictating its targeting to the medial cortex during cytokinesis are unknown. In this paper, we define a short motif within the N terminus of Cdc12 that binds directly to the F-BAR domain of the scaffolding protein Cdc15. Mutations preventing the Cdc12–Cdc15 interaction resulted in reduced Cdc12, F-actin, and actin-binding proteins at the CR, which in turn led to a delay in CR formation and sensitivity to other perturbations of CR assembly. We conclude that Cdc15 contributes to CR formation and cytokinesis via formin Cdc12 recruitment, defining a novel cytokinetic function for an F-BAR domain.     

Sorting nexin 9 interacts with dynamin 1 and N-WASP and coordinates synaptic vesicle endocytosis

Sorting nexin 9 (SNX9) is a member of the sorting nexin family of proteins, each of which contains a characteristic Phox homology domain. SNX9 is widely expressed and plays a role in clathrin-mediated endocytosis, but it is not known if it is present in neuronal cells. We report that SNX9 is expressed in the presynaptic compartment of cultured hippocampal neurons, where it binds to dynamin-1 and N-WASP. Overexpression of full-length SNX9 or a C-terminal truncated version caused severe defects in synaptic vesicle endocytosis during, as well as after, stimulation. Knockdown of SNX9 with short interfering RNA also reduced synaptic vesicle endocytosis, and the W39A mutation of SNX9 abolished the inhibitory effect of SNX9 on endocytosis. Rescue experiments showed that most of the effect of SNX9 on endocytosis results from its interaction with dynamin 1, although its interaction with N-WASP contributes in some degree. We further showed that SNX9 dimerizes through its C-terminal domain, suggesting that it may interact simultaneously with dynamin 1 and N-WASP. We propose that SNX9 interacts with dynamin-1 and N-WASP in presynaptic terminals, where it links actin dynamics and synaptic vesicle endocytosis.     

A Dictyostelium homologue of WASP is required for polarized F-actin assembly during chemotaxis

The actin cytoskeleton controls the overall structure of cells and is highly polarized in chemotaxing cells, with F-actin assembled predominantly in the anterior leading edge and to a lesser degree in the cell's posterior. Wiskott-Aldrich syndrome protein (WASP) has emerged as a central player in controlling actin polymerization. We have investigated WASP function and its regulation in chemotaxing Dictyostelium cells and demonstrated the specific and essential role of WASP in organizing polarized F-actin assembly in chemotaxing cells. Cells expressing very low levels of WASP show reduced F-actin levels and significant defects in polarized F-actin assembly, resulting in an inability to establish axial polarity during chemotaxis. GFP-WASP preferentially localizes at the leading edge and uropod of chemotaxing cells and the B domain of WASP is required for the localization of WASP. We demonstrated that the B domain binds to PI(4,5)P2 and PI(3,4,5)P3 with similar affinities. The interaction between the B domain and PI(3,4,5)P3 plays an important role for the localization of WASP to the leading edge in chemotaxing cells. Our results suggest that the spatial and temporal control of WASP localization and activation is essential for the regulation of directional motility.     

The structural basis of actin interaction with multiple WH2/beta-thymosin motif-containing proteins

Participation of actin in cellular processes relies on the dynamics of filament assembly. Filament elongation is fed by monomeric actin in complex with either profilin or a Wiscott-Aldrich syndrome protein (WASP) homology domain 2 (WH2)/beta-thymosin (betaT) domain. WH2/betaT motif repetition (typified by ciboulot) or combination with nonrelated domains (as found in N-WASP) results in proteins that yield their actin to filament elongation. Here, we report the crystal structures of actin bound hybrid proteins, constructed between gelsolin and WH2/betaT domains from ciboulot or N-WASP. We observe the C-terminal half of ciboulot domain 2 bound to actin. In solution, we show that cibolout domains 2 and 3 bind to both G- and F-actin, and that whole ciboulot forms a complex with two actin monomers. In contrast, the analogous portion of N-WASP WH2 domain 2 is detached from actin, indicating that the C-terminal halves of the betaT and WH2 motifs are not functionally analogous.     

Nck and phosphatidylinositol 4,5-bisphosphate synergistically activate actin polymerization through the N-WASP-Arp2/3 pathway

The Wiskott-Aldrich syndrome protein (WASP) and its relative neural WASP (N-WASP) regulate the nucleation of actin filaments through their interaction with the Arp2/3 complex and are regulated in turn by binding to GTP-bound Cdc42 and phosphatidylinositol 4,5-bisphosphate. The Nck Src homology (SH) 2/3 adaptor binds via its SH3 domains to a proline-rich region on WASP and N-WASP and has been implicated in recruitment of these proteins to sites of tyrosine phosphorylation. We show here that Nck SH3 domains dramatically stimulate the rate of nucleation of actin filaments by purified N-WASP in the presence of Arp2/3 in vitro. All three Nck SH3 domains are required for maximal activation. Nck-stimulated actin nucleation by N-WASP.Arp2/3 complexes is further stimulated by phosphatidylinositol 4,5-bisphosphate, but not by GTP-Cdc42, suggesting that Nck and Cdc42 activate N-WASP by redundant mechanisms. These results suggest the existence of an Nck-dependent, Cdc42-independent mechanism to induce actin polymerization at tyrosine-phosphorylated Nck binding sites.     

Wsp1, a GBD/CRIB domain-containing WASP homolog, is required for growth, morphogenesis, and virulence of Cryptococcus neoformans

Human endocytic protein ITSN1 regulates actin reorganization by activating Rho family GTPases, such as Cdc42. The process is enhanced by ITSN binding of WASP, an effector of Cdc42 and a potent activator of actin polymerization. In the human pathogen Cryptococcus neoformans, endocytic protein Cin1 also interacts with Cdc42 and Wsp1, an uncharacterized WASP homolog, but the significance of these interactions remains unknown. Wsp1 contains several conserved domains, including a WASP homology 1 domain (WH1), a GTPase binding/Cdc42 and Rac interactive binding domain (GBD/CRIB), and a C-terminal domain composed of verprolin-like, central, and acidic motifs (VCA). Thus, Wsp1 exhibits domain compositions more similar to human WASP proteins than Saccharomyces cerevisiae Las17/Bee1, a WASP homolog lacking the GDB/CRIB domain. Wsp1 is not an essential protein; however, the wsp1 mutant exhibited defects in growth, cytokinesis, chitin distribution, and endocytosis and exocytosis. The wsp1 mutant was also unable to undergo genetic cross, produce the polysaccharide capsule, or secrete the enzyme urease. An in vitro phagocytosis assay showed a higher phagocytic index for the wsp1 mutant, whose ability to cause lethal infection in a murine model of cryptococcosis was also attenuated. Our studies reveal divergent evolution of WASP proteins in the fungal phylum and suggest that the conserved function of WASP proteins in the actin cytoskeleton may also impact fungal virulence.     

SNX9 regulates dynamin assembly and is required for efficient clathrin-mediated endocytosis

Dynamin, a central player in clathrin-mediated endocytosis, interacts with several functionally diverse SH3 domain-containing proteins. However, the role of these interactions with regard to dynamin function is poorly defined. We have investigated a recently identified protein partner of dynamin, SNX9, sorting nexin 9. SNX9 binds directly to both dynamin-1 and dynamin-2. Moreover by stimulating dynamin assembly, SNX9 stimulates dynamin's basal GTPase activity and potentiates assembly-stimulated GTPase activity on liposomes. In fixed cells, we observe that SNX9 partially localizes to clathrin-coated pits. Using total internal reflection fluorescence microscopy in living cells, we detect a transient burst of EGFP-SNX9 recruitment to clathrin-coated pits that occurs during the late stages of vesicle formation and coincides spatially and temporally with a burst of dynamin-mRFP fluorescence. Transferrin internalization is inhibited in HeLa cells after siRNA-mediated knockdown of SNX9. Thus, our results establish that SNX9 is required for efficient clathrin-mediated endocytosis and suggest that it functions to regulate dynamin activity.     

Three different actin filament assemblies occur in every hair cell: each contains a specific actin crosslinking protein

The apex of hair cells of the chicken auditory organ contains three different kinds of assemblies of actin filaments in close spatial proximity. These are (a) paracrystals of actin filaments with identical polarity in stereocilia, (b) a dense gellike meshwork of actin filaments forming the cuticular plate, and (c) a bundle of parallel actin filaments with mixed polarities that constitute the circumferential filament belt attached to the cytoplasmic aspect of the zonula adhaerens (ZA). Each different supramolecular assembly of actin filaments contains a specific actin filament cross-linking protein which is unique to that particular assembly. Thus fimbrin appears to be responsible for paracrystallin packing of actin filaments in stereocillia; an isoform of spectrin resides in the cuticular plate where it forms the whisker-like crossbridges, and alpha actinin is the actin crosslinking protein of the circumferential ZA bundle. Tropomyosin, which stabilizes actin filaments, is present in all the actin filament assemblies except for the stereocilia. Another striking finding was that myosin appears to be absent from the ZA ring and cuticular plate of hair cells although present in the ZA ring of supporting cells. The abundance of myosin in the ZA ring of the surrounding supporting cells means that it may be important in forming a supporting tensile cellular framework in which the hair cells are inserted.     

Different WASP family proteins stimulate different Arp2/3 complex-dependent actin-nucleating activities

Background: Assembly and organization of actin filaments are required for many cellular processes, including locomotion and division. In many cases, actin assembly is initiated when proteins of the WASP/Scar family respond to signals from Rho family G proteins and stimulate the actin-nucleating activity of the Arp2/3 complex. Two questions of fundamental importance raised in the study of actin dynamics concern the molecular mechanism of Arp2/3-dependent actin nucleation and how different signaling pathways that activate the same Arp2/3 complex produce actin networks with different three-dimensional architectures?Results: We directly compared the activity of the Arp2/3 complex in the presence of saturating concentrations of the minimal Arp2/3-activating domains of WASP, N-WASP, and Scar1 and found that each induces unique kinetics of actin assembly. In cell extracts, N-WASP induces rapid actin polymerization, while Scar1 fails to induce detectable polymerization. Using purified proteins, Scar1 induces the slowest rate of nucleation. WASP activity is 16-fold higher, and N-WASP activity is 70-fold higher. The data for all activators fit a mathematical model in which one activated Arp2/3 complex, one actin monomer, and an actin filament combine into a preactivation complex which then undergoes a first-order activation step to become a nucleus. The differences between Scar and N-WASP activity are explained by differences in the rate constants for the activation step. Changing the number of actin binding sites on a WASP family protein, either by removing a WH2 domain from N-WASP or by adding WH2 domains to Scar1, has no significant effect on nucleation activity. The addition of a three amino acid insertion found in the C-terminal acidic domains of WASP and N-WASP, however, increases the activity of Scar1 by more than 20-fold. Using chemical crosslinking assays, we determined that both N-WASP and Scar1 induce a conformational change in the Arp2/3 complex but crosslink with different efficiencies to the small molecular weight subunits p18 and p14.Conclusion: The WA domains of N-WASP, WASP, and Scar1 bind actin and Arp2/3 with nearly identical affinities but stimulate rates of actin nucleation that vary by almost 100-fold. The differences in nucleation rate are caused by differences in the number of acidic amino acids at the C terminus, so each protein is tuned to produce a different rate of actin filament formation. Arp2/3, therefore, is not regulated by a simple on-off switch. Precise tuning of the filament formation rate may help determine the architecture of actin networks produced by different nucleation-promoting factors.     

Immunocytochemical detection of calcium-binding protein in the cochlear and vestibular hair cells of the rat

Specific antibodies raised against human cerebellar calcium-binding protein (CaBP) intensely labelled the cochlear hair cells of the rat. The vestibular hair cells also stained weakly. In both inner and outer cochlear hair cells, the cuticular plate was the most stained area. These results suggest that CaBP may prevent excessive concentrations of intracellular calcium and thus modulate some Ca2+-mediated biochemical processes, especially at the level of the cuticular plate and stereocilia; CaBP could be involved in the mechanochemical coupling of hearing or vestibular function.     

WASP-interacting protein is important for actin filament elongation and prompt pseudopod formation in response to a dynamic chemoattractant gradient

The role of WASP-interacting protein (WIP) in the process of F-actin assembly during chemotaxis of Dictyostelium was examined. Mutations of the WH1 domain of WASP led to a reduction in binding to WIPa, a newly identified homolog of mammalian WIP, a reduction of F-actin polymerization at the leading edge, and a reduction in chemotactic efficiency. WIPa localizes to sites of new pseudopod protrusion and colocalizes with WASP at the leading edge. WIPa increases F-actin elongation in vivo and in vitro in a WASP-dependent manner. WIPa translocates to the cortical membrane upon uniform cAMP stimulation in a time course that parallels F-actin polymerization. WIPa-overexpressing cells exhibit multiple microspike formation and defects in chemotactic efficiency due to frequent changes of direction. Reduced expression of WIPa by expressing a hairpin WIPa (hp WIPa) construct resulted in more polarized cells that exhibit a delayed response to a new chemoattractant source due to delayed extension of pseudopod toward the new gradient. These results suggest that WIPa is required for new pseudopod protrusion and prompt reorientation of cells toward a new gradient by initiating localized bursts of actin polymerization and/or elongation.     

The SH3 domains of two PCH family members cooperate in assembly of the Schizosaccharomyces pombe contractile ring

Schizosaccharomyces pombe cdc15 homology (PCH) family members participate in many cellular processes by bridging the plasma membrane and cytoskeleton. Their F-BAR domains bind and curve membranes, whereas other domains, typically SH3 domains, are expected to provide cytoskeletal links. We tested this prevailing model of functional division in the founding member of the family, Cdc15, which is essential for cytokinesis in S. pombe, and in the related PCH protein, Imp2. We find that the distinct functions of Imp2 and Cdc15 are SH3 domain independent. However, the Cdc15 and Imp2 SH3 domains share an essential role in recruiting proteins to the contractile ring, including Pxl1 and Fic1. Together, Pxl1 and Fic1, a previously uncharacterized C2 domain protein, add structural integrity to the contractile ring and prevent it from fragmenting during division. Our data indicate that the F-BAR proteins Cdc15 and Imp2 contribute to a single biological process with both distinct and overlapping functions.     

Actin filaments, stereocilia and hair cells of the bird cochlea. VI. How the number and arrangement of stereocilia are determined.

Beginning in 8-day embryos, stereocilia sprout from the apical surface of hair cells apparently at random. As the embryo continues to develop, the number of stereocilia increases. By 10 1/2 days the number is approximately the same as that encountered extending from mature hair cells at the same relative positions in the adult cochlea. Surprisingly, over the next 2-3 days the number of stereocilia continues to increase so that hair cells in a 12-day embryo have 1 1/2 to 2 times as many stereocilia as in adult hair cells. In short, there is an overshoot in stereociliary number. During the same period in which stereocilia are formed (9-12 days) the apical surface of each hair cell is filled with closely packed stereocilia; thus the surface area is proportional to the number of stereocilia present per hair cell, as if these features were coupled. The staircase begins to form in a 10-day embryo, with what will be the tallest row beginning to elongate first and gradually row after row begins to elongate by incorporation of stereocilia at the foot of the staircase. Extracellular connections or tip linkages appear as the stereocilia become incorporated into the staircase. After a diminutive staircase has formed, eg. in a 12-day embryo, the remaining stereocilia located at the foot of the staircase begin to be reabsorbed, a process that occurs during the next few days. We conclude that the hair cell determines the number of stereocilia to form by filling up the available apical surface area with stereocilia and then, by cropping back those that are not stabilized by extracellular linkages, arrives at the appropriate number. Furthermore, the stereociliary pattern, which changes from having a round cross-sectional profile to a rectangular one, is generated by these same linkages which lock the stereocilia into a precise pattern. As this pattern is established, we envision that the stereocilia flow over the apical surface until frozen in place by the formation of the cuticular plate in the apical cell cytoplasm.