Pieta K. Mattila

Molecular Mechanisms of Membrane Deformation by I-BAR Domain Proteins

BACKGROUND Generation of membrane curvature is critical for the formation of plasma membrane protrusions and invaginations and for shaping intracellular organelles. Among the central regulators of membrane dynamics are the BAR superfamily domains, which deform membranes into tubular structures. In contrast to the relatively well characterized BAR and F-BAR domains that promote the formation of plasma membrane invaginations, I-BAR domains induce plasma membrane protrusions through a poorly understood mechanism. RESULTS We show that I-BAR domains induce strong PI(4,5)P(2) clustering upon membrane binding, bend the membrane through electrostatic interactions, and remain dynamically associated with the inner leaflet of membrane tubules. Thus, I-BAR domains induce the formation of dynamic membrane protrusions to the opposite direction than do BAR and F-BAR domains. Strikingly, comparison of different I-BAR domains revealed that they deform PI(4,5)P(2)-rich membranes through distinct mechanisms. IRSp53 and IRTKS I-BARs bind membranes mainly through electrostatic interactions, whereas MIM and ABBA I-BARs additionally insert an amphipathic helix into the membrane bilayer, resulting in larger tubule diameter in vitro and more efficient filopodia formation in vivo. Furthermore, FRAP analysis revealed that whereas the mammalian I-BAR domains display dynamic association with filopodia, the C. elegans I-BAR domain forms relatively stable structures inside the plasma membrane protrusions. CONCLUSIONS These data define I-BAR domain as a functional member of the BAR domain superfamily and unravel the mechanisms by which I-BAR domains deform membranes to induce filopodia in cells. Furthermore, our work reveals unexpected divergence in the mechanisms by which evolutionarily distinct groups of I-BAR domains interact with PI(4,5)P(2)-rich membranes.

WH2 domain: a small, versatile adapter for actin monomers.

The actin cytoskeleton plays a central role in many cell biological processes. The structure and dynamics of the actin cytoskeleton are regulated by numerous actin‐binding proteins that usually contain one of the few known actin‐binding motifs. WH2 domain ( ASP omology domain‐ ) is a ∼35 residue actin monomer‐binding motif, that is found in many different regulators of the actin cytoskeleton, including the β‐thymosins, ciboulot, WASP ( iskott ldrich yndrome rotein), verprolin/WIP ( ASP‐ nteracting rotein), Srv2/CAP (adenylyl yclase‐ ssociated rotein) and several uncharacterized proteins. The most highly conserved residues in the WH2 domain are important in β‐thymosins interactions with actin monomers, suggesting that all WH2 domains may interact with actin monomers through similar interfaces. Our sequence database searches did not reveal any WH2 domain‐containing proteins in plants. However, we found three classes of these proteins: WASP, Srv2/CAP and verprolin/WIP in yeast and animals. This suggests that the WH2 domain is an ancient actin monomer‐binding motif that existed before the divergence of fungal and animal lineages.

Contractility-dependent actin dynamics in cardiomyocyte sarcomeres

In contrast to the highly dynamic actin cytoskeleton in non-muscle cells, actin filaments in muscle sarcomeres are thought to be relatively stable and undergo dynamics only at their ends. However, many proteins that promote rapid actin dynamics are also expressed in striated muscles. We show that a subset of actin filaments in cardiomyocyte sarcomeres displays rapid turnover. Importantly, we found that turnover of these filaments depends on contractility of the cardiomyocytes. Studies using an actin-polymerization inhibitor suggest that the pool of dynamic actin filaments is composed of filaments that do not contribute to contractility. Furthermore, we provide evidence that ADF/cofilins, together with myosin-induced contractility, are required to disassemble non-productive filaments in developing cardiomyocytes. These data indicate that an excess of actin filaments is produced during sarcomere assembly, and that contractility is applied to recognize non-productive filaments that are subsequently destined for depolymerization. Consequently, contractility-induced actin dynamics plays an important role in sarcomere maturation.

Twinfilin is required for actin-dependent developmental processes in Drosophila

The actin cytoskeleton is essential for cellular remodeling and many developmental and morphological processes. Twinfilin is a ubiquitous actin monomer–binding protein whose biological function has remained unclear. We discovered and cloned the Drosophila twinfilin homologue, and show that this protein is ubiquitously expressed in different tissues and developmental stages. A mutation in the twf gene leads to a number of developmental defects, including aberrant bristle morphology. This results from uncontrolled polymerization of actin filaments and misorientation of actin bundles in developing bristles. In wild-type bristles, twinfilin localizes diffusively to cytoplasm and to the ends of actin bundles, and may therefore be involved in localization of actin monomers in cells. We also show that twinfilin and the ADF/cofilin encoding gene twinstar interact genetically in bristle morphogenesis. These results demonstrate that the accurate regulation of size and dynamics of the actin monomer pool by twinfilin is essential for a number of actin-dependent developmental processes in multicellular eukaryotes.

Mechanism and biological role of profilin-Srv2/CAP interaction

Profilin and cyclase-associated protein (CAP, known in yeast as Srv2) are ubiquitous and abundant actin monomer-binding proteins. Profilin catalyses the nucleotide exchange on actin monomers and promotes their addition to filament barbed ends. Srv2/CAP recycles newly depolymerized actin monomers from ADF/cofilin for subsequent rounds of polymerization. Srv2/CAP also harbors two proline-rich motifs and has been suggested to interact with profilin. However, the mechanism and biological role of the possible profilin-Srv2/CAP interaction has not been investigated. Here, we show that Saccharomyces cerevisiae Srv2 and profilin interact directly (KD ∼1.3 μM) and demonstrate that a specific proline-rich motif in Srv2 mediates this interaction in vitro and in vivo. ADP-actin monomers and profilin do not interfere with each others binding to Srv2, suggesting that these three proteins can form a ternary complex. Genetic and cell biological analyses on an Srv2 allele (srv2-201) defective in binding profilin reveals that a direct interaction with profilin is not essential for Srv2 cellular function. However, srv2-201 causes a moderate increase in cell size and partially suppresses the cell growth and actin organization defects of an actin binding mutant profilin (pfy1-4). Together these data suggest that Srv2 is an important physiological interaction partner of profilin.

Missing-in-metastasis MIM/MTSS1 promotes actin assembly at intercellular junctions and is required for integrity of kidney epithelia.

MIM/MTSS1 is a tissue-specific regulator of plasma membrane dynamics, whose altered expression levels have been linked to cancer metastasis. MIM deforms phosphoinositide-rich membranes through its I-BAR domain and interacts with actin monomers through its WH2 domain. Recent work proposed that MIM also potentiates Sonic hedgehog (Shh)-induced gene expression. Here, we generated MIM mutant mice and found that full-length MIM protein is dispensable for embryonic development. However, MIM-deficient mice displayed a severe urinary concentration defect caused by compromised integrity of kidney epithelia intercellular junctions, which led to bone abnormalities and end-stage renal failure. In cultured kidney epithelial (MDCK) cells, MIM displayed dynamic localization to adherens junctions, where it promoted Arp2/3-mediated actin filament assembly. This activity was dependent on the ability of MIM to interact with both membranes and actin monomers. Furthermore, results from the mouse model and cell culture experiments suggest that full-length MIM is not crucial for Shh signaling, at least during embryogenesis. Collectively, these data demonstrate that MIM modulates interplay between the actin cytoskeleton and plasma membrane to promote the maintenance of intercellular contacts in kidney epithelia.

ABBA regulates plasma-membrane and actin dynamics to promote radial glia extension

Radial glia play key roles in neuronal migration, axon guidance, and neurogenesis during development of the central nervous system. However, the molecular mechanisms regulating growth and morphology of these extended cells are unknown. We show that ABBA, a novel member of the IRSp53-MIM protein family, is enriched in different types of radial glia. ABBA binds ATP-actin monomers with high affinity and deforms PtdIns(4,5)P2-rich membranes in vitro through its WH2 and IM domains, respectively. In radial-glia-like C6-R cells, ABBA localises to the interface between the actin cytoskeleton and plasma membrane, and its depletion by RNAi led to defects in lamellipodial dynamics and process extension. Together, this study identifies ABBA as a novel regulator of actin and plasma membrane dynamics in radial glial cells, and provides evidence that membrane binding and deformation activity is critical for the cellular functions of IRSp53-MIM-ABBA family proteins.

MIM-Induced Membrane Bending Promotes Dendritic Spine Initiation

Proper morphogenesis of neuronal dendritic spines is essential for the formation of functional synaptic networks. However, it is not known how spines are initiated. Here, we identify the inverse-BAR (I-BAR) protein MIM/MTSS1 as a nucleator of dendritic spines. MIM accumulated to future spine initiation sites in a PIP2-dependent manner and deformed the plasma membrane outward into a proto-protrusion via its I-BAR domain. Unexpectedly, the initial protrusion formation did not involve actin polymerization. However, PIP2-dependent activation of Arp2/3-mediated actin assembly was required for protrusion elongation. Overexpression of MIM increased the density of dendritic protrusions and suppressed spine maturation. In contrast, MIM deficiency led to decreased density of dendritic protrusions and larger spine heads. Moreover, MIM-deficient mice displayed altered glutamatergic synaptic transmission and compatible behavioral defects. Collectively, our data identify an important morphogenetic pathway, which initiates spine protrusions by coupling phosphoinositide signaling, direct membrane bending, and actin assembly to ensure proper synaptogenesis.

Reply to: Are β-thymosins WH2 domains?

WH2 (WASP homology 2) domain is a ubiquitous actin monomer binding motif that is present in a wide variety of regulators of the actin cytoskeleton. The article by John Edwards presents an analysis suggesting that b-thymosins (including proteins containing tandem b-thymosin-like repeats) and other WH2 domains would not have a common ancestry. As already pointed out in our review article [1], the WH2 domain is a very short protein motif and thus reliable phylogenetic analysis of this domain is difficult. Thus, we agree that it is not possible to conclude whether b-thymosins and other WH2 domains have a common ancestor. However, all available biochemical and structural data indicate that b-thymosins and other WH2 domains belong, from a structural and functional point of view, to the same family of actin-binding motifs. Alignments of b-thymosins and other WH2 domains show that their most highly conserved regions are the LKK motif and region N-terminal of it. In contrast, the C-terminal regions of b-thymosins display high sequence identity to each other, whereas the C-terminal regions of other WH2 domains are very heterogeneous [1,2]. Recent structural studies on b-thymosin and ciboulot provide a plausible explanation for the conservation of these sequence features. The most critical actin-binding region is located in the LKK motif and in the a-helical region preceding this motif. These regions interact with the subdomains 1 and 3 at the ‘barbed-end’ of the actin monomer. The highly conserved C-terminal region of b-thymosins forms an additional a-helix, which interacts with the subdomain 2 at the ‘pointed-end’ of actin monomer. This C-terminal helix is present in actin filament sequestering b-thymosins but it is absent from ciboulot, which promotes actin filament assembly. Interestingly, b-thymosin can be changed from an actin filament sequestering to assembly promoting protein by disrupting this a-helix by site-directed mutagenesis [3,4]. Together, these data provide a plausible explanation for the lack of sequence conservation at the C-terminal regions of those WH2 domains that do not display actin monomer sequestering activity. Consequently, sequence conservation between b-thymosins and other WH2 domains is expected to be limited in the N-terminal actin-binding region.