Parallel actin bundles and their multiple actin-bundling proteins

Parallel actin bundles are present in a diverse array of structures, where they are critical determinants of cellular shape and physiology. In the past 18 months, new findings have solidified the concept that parallel actin bundles are assembled in cells through the sequential action of multiple actin-bundling proteins and have begun to shed light on the roles played by the individual actin-bundling proteins.     

A new role for the architecture of microvillar actin bundles in apical retention of membrane proteins

Actin-bundling proteins are identified as key players in the morphogenesis of thin membrane protrusions. Until now, functional redundancy among the actin-bundling proteins villin, espin, and plastin-1 has prevented definitive conclusions regarding their role in intestinal microvilli. We report that triple knockout mice lacking these microvillar actin-bundling proteins suffer from growth delay but surprisingly still develop microvilli. However, the microvillar actin filaments are sparse and lack the characteristic organization of bundles. This correlates with a highly inefficient apical retention of enzymes and transporters that accumulate in subapical endocytic compartments. Myosin-1a, a motor involved in the anchorage of membrane proteins in microvilli, is also mislocalized. These findings illustrate, in vivo, a precise role for local actin filament architecture in the stabilization of apical cargoes into microvilli. Hence, the function of actin-bundling proteins is not to enable microvillar protrusion, as has been assumed, but to confer the appropriate actin organization for the apical retention of proteins essential for normal intestinal physiology.     

Dimerization and actin-bundling properties of villin and its role in the assembly of epithelial cell brush borders

Villin is a major actin-bundling protein in the brush border of epithelial cells. In this study we demonstrate for the first time that villin can bundle actin filaments using a single F-actin binding site, because it has the ability to self-associate. Using fluorescence resonance energy transfer, we demonstrate villin self-association in living cells in microvilli and in growth factor-stimulated cells in membrane ruffles and lamellipodia. Using sucrose density gradient, size-exclusion chromatography, and matrix-assisted laser desorption ionization time-of-flight, the majority of villin was identified as a monomer or dimer. Villin dimers were also identified in Caco-2 cells, which endogenously express villin and Madin-Darby canine kidney cells that ectopically express villin. Using truncation mutants of villin, site-directed mutagenesis, and fluorescence resonance energy transfer, an amino-terminal dimerization site was identified that regulated villin self-association in parallel conformation as well as actin bundling by villin. This detailed analysis describes for the first time microvillus assembly by villin, redefines the actin-bundling function of villin, and provides a molecular mechanism for actin bundling by villin, which could have wider implications for other actin cross-linking proteins that share a villin-like headpiece domain. Our study also provides a molecular basis to separate the morphologically distinct actin-severing and actin-bundling properties of villin.     

Interaction of two actin-binding proteins,synaptopodin and alpha-actinin-4, with the tight junction protein MAGI-1

In an attempt to find podocyte-expressed proteins that may interact with the tight junction protein MAGI-1, we screened a glomerulus-enriched cDNA library with a probe consisting of both WW domains of MAGI-1. One of the isolated clones contained two WW domain-binding motifs and was identified as a portion of the actin-bundling protein synaptopodin. In vitro binding assays confirmed this interaction between MAGI-1 and synaptopodin and identified the second WW domain of MAGI-1 to be responsible for the interaction. MAGI-1 and synaptopodin can also interact in vivo, as they can be immunoprecipitated together from HEK293 cell lysates. Another actin-bundling protein that is found in glomerular podocytes and shown to be mutated in an inheritable form of glomerulosclerosis is alpha-actinin-4. We show that alpha-actinin-4 is also capable of binding to MAGI-1 in in vitro binding assays and that this interaction is mediated by the fifth PDZ domain of MAGI-1 binding to the C terminus of alpha-actinin-4. Exogenously expressed synaptopodin and alpha-actinin-4 were found to colocalize along with endogenous MAGI-1 at the tight junction of Madin-Darby canine kidney cells. The interaction and colocalization of MAGI-1 with two actin-bundling proteins suggest that MAGI-1 may play a role in actin cytoskeleton dynamics within polarized epithelial cells.     

Microglia/macrophage-specific protein Iba1 binds to fimbrin and enhances its actin-bundling activity

Ionized calcium binding adaptor molecule 1 (Iba1) is a microglia/macrophage-specific calcium-binding protein. Iba1 has the actin-bundling activity and participates in membrane ruffling and phagocytosis in activated microglia. In order to understand the Iba1-related intracellular signalling pathway in greater detail, we employed a yeast two-hybrid screen to isolate an Iba1-interacting molecule and identified another actin-bundling protein, L-fimbrin. In response to stimulation, L-fimbrin accumulated and co-localized with Iba1 in membrane ruffles induced by M-CSF-stimulation and phagocytic cups formed by IgG-opsonized beads in microglial cell line MG5. L-fimbrin was shown to associate with Iba1 in cell lysate of COS-7 expressing L-fimbrin and Iba1. By using purified proteins, direct binding of Iba1 to L-fimbrin was demonstrated by immunoprecipitation, glutathione S-transferase pull-down assays and ligand overlay assays. The binding of Iba1 was also found to increase the actin-bundling activity of L-fimbrin. These results indicate that Iba1 forms complexes with L-fimbrin in membrane ruffles and phagocytic cups, and suggest that Iba1 co-operates with L-fimbrin in modulating actin reorganization to facilitate cell migration and phagocytosis by microglia.     

The deaf jerker mouse has a mutation in the gene encoding the espin actin-bundling proteins of hair cell stereocilia and lacks espins

The espins are actin-bundling proteins of brush border microvilli and Sertoli cell-spermatid junctions. We have determined that espins are also present in hair cell stereocilia and have uncovered a connection between the espin gene and jerker, a recessive mutation that causes hair cell degeneration, deafness, and vestibular dysfunction. The espin gene maps to the same region of mouse chromosome 4 as jerker. The tissues of jerker mice do not accumulate espin proteins but contain normal levels of espin mRNAs. The espin gene of jerker mice has a frameshift mutation that affects the espin C-terminal actin-bundling module. These data suggest that jerker mice are, in effect, espin null and that the jerker phenotype results from a mutation in the espin gene.     

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.     

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.     

Domain analysis of cortexillin I: actin-bundling, PIP(2)-binding and the rescue of cytokinesis

Cortexillins are actin-bundling proteins that form a parallel two-stranded coiled-coil rod. Actin-binding domains of the alpha-actinin/spectrin type are located N-terminal to the rod and unique sequence elements are found in the C-terminal region. Domain analysis in vitro revealed that the N-terminal domains are not responsible for the strong actin-filament bundling activity of cortexillin I. The strongest activity resides in the C-terminal region. Phosphatidylinositol 4,5-bisphosphate (PIP(2)) suppresses this bundling activity by binding to a C-terminal nonapeptide sequence. These data define a new PIP(2)-regulated actin-bundling site. In vivo the PIP(2)-binding motif enhances localization of a C-terminal cortexillin I fragment to the cell cortex and improves the rescue of cytokinesis. This motif is not required, however, for translocation to the cleavage furrow. A model is presented proposing that cortexillin translocation is based on a mitotic cycle of polar actin polymerization and midzone depolymerization.     

Roles of the espin actin-bundling proteins in the morphogenesis and stabilization of hair cell stereocilia revealed in CBA/CaJ congenic jerker mice

Hearing and vestibular function depend on mechanosensory staircase collections of hair cell stereocilia, which are produced from microvillus-like precursors as their parallel actin bundle scaffolds increase in diameter and elongate or shorten. Hair cell stereocilia contain multiple classes of actin-bundling protein, but little is known about what each class contributes. To investigate the roles of the espin class of actin-bundling protein, we used a genetic approach that benefited from a judicious selection of mouse background strain and an examination of the effects of heterozygosity. A congenic jerker mouse line was prepared by repeated backcrossing into the inbred CBA/CaJ strain, which is known for excellent hearing and minimal age-related hearing loss. We compared stereocilia in wild-type CBA/CaJ mice, jerker homozygotes that lack espin proteins owing to a frameshift mutation in the espin gene, and jerker heterozygotes that contain reduced espin levels. The lack of espins radically impaired stereociliary morphogenesis, resulting in stereocilia that were abnormally thin and short, with reduced differential elongation to form a staircase. Mean stereociliary diameter did not increase beyond ～0.10-0.14 μm, making stereocilia ～30%-60% thinner than wild type and suggesting that they contained ～50%-85% fewer actin filaments. These characteristics indicate a requirement for espins in the appositional growth and differential elongation of the stereociliary parallel actin bundle and fit the known biological activities of espins in vitro and in transfected cells. The stereocilia of jerker heterozygotes showed a transient proximal-distal tapering suggestive of haploinsufficiency and a slowing of morphogenesis that revealed previously unrecognized assembly steps and intermediates. The lack of espins also led to a region-dependent degeneration of stereocilia involving shortening and collapse. We conclude that the espin actin-bundling proteins are required for the assembly and stabilization of the stereociliary parallel actin bundle.     

Espin cross-links cause the elongation of microvillus-type parallel actin bundles in vivo

The espin actin-bundling proteins, which are the target of the jerker deafness mutation, caused a dramatic, concentration-dependent lengthening of LLC-PK1-CL4 cell microvilli and their parallel actin bundles. Espin level was also positively correlated with stereocilium length in hair cells. Villin, but not fascin or fimbrin, also produced noticeable lengthening. The espin COOH-terminal peptide, which contains the actin-bundling module, was necessary and sufficient for lengthening. Lengthening was blocked by 100 nM cytochalasin D. Espin cross-links slowed actin depolymerization in vitro less than twofold. Elimination of an actin monomer-binding WASP homology 2 domain and a profilin-binding proline-rich domain from espin did not decrease lengthening, but made it possible to demonstrate that actin incorporation was restricted to the microvillar tip and that bundles continued to undergo actin treadmilling at approximately 1.5 s-1 during and after lengthening. Thus, through relatively subtle effects on actin polymerization/depolymerization reactions in a treadmilling parallel actin bundle, espin cross-links cause pronounced barbed-end elongation and, thereby, make a longer bundle without joining shorter modules.     

Dual actin-bundling and protein kinase C-binding activities of fascin regulate carcinoma cell migration downstream of Rac and contribute to metastasis

Recurrence of carcinomas due to cells that migrate away from the primary tumor is a major problem in cancer treatment. Immunohistochemical analyses of human carcinomas have consistently correlated up-regulation of the actin-bundling protein fascin with a clinically aggressive phenotype and poor prognosis. To understand the functional and mechanistic contributions of fascin, we undertook inducible short hairpin RNA (shRNA) knockdown of fascin in human colon carcinoma cells derived from an aggressive primary tumor. Fascin-depletion led to decreased numbers of filopodia and altered morphology of cell protrusions, decreased Rac-dependent migration on laminin, decreased turnover of focal adhesions, and, in vivo, decreased xenograft tumor development and metastasis. cDNA rescue of fascin shRNA-knockdown cells with wild-type green fluorescent protein-fascin or fascins mutated at the protein kinase C (PKC) phosphorylation site revealed that both the actin-bundling and active PKC-binding activities of fascin are required for the organization of filopodial protrusions, Rac-dependent migration, and tumor metastasis. Thus, fascin contributes to carcinoma migration and metastasis through dual pathways that impact on multiple subcellular structures needed for cell migration.     

Elongation factor 1α is a component of the subcortical actin bundles of characean algae.

Antibodies to elongation factor 1α (EF1α), a known 50 kDa actin-bundling protein in Dictyostelium, identified a protein in a whole cell extract of the characean alga Nitella pseudoflabellata that had an apparent molecular weight of 51 kDa. Indirect immunofluorescence microscopy revealed labelling by the EF1α antibodies of the subcortical actin bundles, even after the motile organelles of the endoplasm were removed by perfusion with ATP-containing solutions.     

Cysteine-rich protein 1 (CRP1) regulates actin filament bundling

Background  

Cysteine-rich protein 1 (CRP1) is a LIM domain containing protein localized to the nucleus and the actin cytoskeleton. CRP1 has been demonstrated to bind the actin-bundling protein α-actinin and proposed to modulate the actin cytoskeleton; however, specific regulatory mechanisms have not been identified.     

Purification of erythrocyte dematin (protein 4.9) reveals an endogenous protein kinase that modulates actin-bundling activity

A partially purified preparation of human erythrocyte protein 4.9, consisting of 48-, 52-, and 55-kilodalton polypeptides, is capable of bundling rabbit muscle actin in vitro (Siegel, D. L., and Branton, D. (1985) J. Cell Biol. 100, 775-785). Purification schemes, peptide mapping, antibody cross-reactivity, and chemical cross-linking techniques show that the 48- and 52-kDa polypeptides are sequence-related phosphorylated components, whereas the 55-kDa polypeptide is not. Purified protein 4.9 (dematin), consisting of 48- and 52-kDa polypeptides, effectively bundles actin in vitro; under similar conditions, the isolated 55-kDa polypeptide does not bundle actin. In fact, when added back to purified dematin, fractions containing the 55-kDa polypeptide can completely abolish dematin's actin-bundling activity. The basis for this inhibitory activity is an endogenous protein kinase that phosporylates both the 48- and 52-kDa isoforms of dematin, thus abolishing dematin's actin-bundling activity (Husain-Chishti, A., Levin, A., and Branton, D. (1988) Nature 334, 718-721). Although the endogenous kinase often co-purifies with the 55-kDa polypeptide, it can be separated from the 55-kDa polypeptide and has the characteristics of a catalytic subunit of a cyclic AMP-dependent protein kinase.     

55-kD actin-bundling protein (p55) is a specific marker for identifying vascular dendritic cells.

Compared with other members of the dendritic cell family, the antigen profile of the recently recognized vascular dendritic cells has received limited attention. This study demonstrates that vascular dendritic cells in the human aorta and carotid arteries express 55-kD actin-bundling protein (p55), a specific marker for blood dendritic cells and Langerhans cells. This finding will facilitate screening of dendritic cells during their isolation from the arterial wall, as well as other investigations.(J Histochem Cytochem 47:1481-1486, 1999)     

Espin contains an additional actin-binding site in its N terminus and is a major actin-bundling protein of the Sertoli cell-spermatid ectoplasmic specialization junctional plaque

The espins are actin-binding and -bundling proteins localized to parallel actin bundles. The 837-amino-acid "espin" of Sertoli cell-spermatid junctions (ectoplasmic specializations) and the 253-amino-acid "small espin" of brush border microvilli are splice isoforms that share a C-terminal 116-amino-acid actin-bundling module but contain different N termini. To investigate the roles of espin and its extended N terminus, we examined the actin-binding and -bundling properties of espin constructs and the stoichiometry and developmental accumulation of espin within the ectoplasmic specialization. An espin construct bound to F-actin with an approximately threefold higher affinity (K(d) = approximately 70 nM) than small espin and was approximately 2.5 times more efficient at forming bundles. The increased affinity appeared to be due to an additional actin-binding site in the N terminus of espin. This additional actin-binding site bound to F-actin with a K(d) of approximately 1 microM, decorated actin stress fiber-like structures in transfected cells, and was mapped to a peptide between the two proline-rich peptides in the N terminus of espin. Espin was detected at approximately 4-5 x 10(6) copies per ectoplasmic specialization, or approximately 1 espin per 20 actin monomers and accumulated there coincident with the formation of parallel actin bundles during spermiogenesis. These results suggest that espin is a major actin-bundling protein of the Sertoli cell-spermatid ectoplasmic specialization.     

Interactions between the yeast SM22 homologue Scp1 and actin demonstrate the importance of actin bundling in endocytosis

The yeast SM22 homologue Scp1 has previously been shown to act as an actin-bundling protein in vitro. In cells, Scp1 localizes to the cortical actin patches that form as part of the invagination process during endocytosis, and its function overlaps with that of the well characterized yeast fimbrin homologue Sac6p. In this work we have used live cell imaging to demonstrate the importance of key residues in the Scp1 actin interface. We have defined two actin binding domains within Scp1 that allow the protein to both bind and bundle actin without the need for dimerization. Green fluorescent protein-tagged mutants of Scp1 also indicate that actin localization does not require the putative phosphorylation site Ser-185 to be functional. Deletion of SCP1 has few discernable effects on cell growth and morphology. However, we reveal that scp1 deletion is compensated for by up-regulation of Sac6. Furthermore, Scp1 levels are increased in the absence of sac6. The presence of compensatory pathways to up-regulate Sac6 or Scp1 levels in the absence of the other suggest that maintenance of sufficient bundling activity is critical within the cell. Analysis of cortical patch assembly and movement during endocytosis reveals a previously undetected role for Scp1 in movement of patches away from the plasma membrane. Additionally, we observe a dramatic increase in patch lifetime in a strain lacking both sac6 and scp1, demonstrating the central role played by actin-bundling proteins in the endocytic process.