Structures of FHOD1-Nesprin1/2 complexes reveal alternate binding modes for the FH3 domain of formins.

The nuclear position in eukaryotes is controlled by a nucleo-cytoskeletal network, critical in cell differentiation, division, and movement. Forces are transmitted through conserved Linker of Nucleoskeleton and Cytoskeleton (LINC) complexes that traverse the nuclear envelope and engage on either side of the membrane with diverse binding partners. Nesprin-2-giant (Nes2G), a LINC element in the outer nuclear membrane, connects to the actin directly as well as through FHOD1, a formin primarily involved in actin bundling. Here, we report the crystal structure of Nes2G bound to FHOD1 and show that the presumed G-binding domain of FHOD1 is rather a spectrin repeat (SR) binding enhancer for the neighboring FH3 domain. The structure reveals that SR binding by FHOD1 is likely not regulated by the diaphanous-autoregulatory domain helix of FHOD1. Finally, we establish that Nes1G also has one FHOD1 binding SR, indicating that these abundant, giant Nesprins have overlapping functions in actin-bundle recruitment for nuclear movement.

Functionally distinct roles for T and Tbx6 during mouse development.

The mouse T-box transcription factors T and Tbx6 are co-expressed in the primitive streak and have unique domains of expression; T is expressed in the notochord, while Tbx6 is expressed in the presomitic mesoderm. T-box factors are related through a shared DNA binding domain, the T-domain, and can therefore bind to similar DNA sequences at least in vitro We investigated the functional similarities and differences of T and Tbx6 DNA binding and transcriptional activity in vitro and their interaction genetically in vivo We show that at one target, Dll1, the T-domains of T and Tbx6 have different affinities for the binding sites present in the mesoderm enhancer. We further show using in vitro assays that T and Tbx6 differentially affect transcription with Tbx6 activating expression tenfold higher than T, that T and Tbx6 can compete at target gene enhancers, and that this competition requires a functional DNA binding domain. Next, we addressed whether T and Tbx6 can compete in vivo First, we generated embryos that express Tbx6 at greater than wild-type levels embryos and show that these embryos have short tails, resembling the T heterozygous phenotype. Next, using the dominant-negative TWis allele, we show that Tbx6+/- TWis/+ embryos share similarities with embryos homozygous for the Tbx6 hypomorphic allele rib-vertebrae, specifically fusions of several ribs and malformation of some vertebrae. Finally, we tested whether Tbx6 can functionally replace T using a knockin approach, which resulted in severe T null-like phenotypes in chimeric embryos generated with ES cells heterozygous for a Tbx6 knockin at the T locus. Altogether, our results of differences in affinity for DNA binding sites and transcriptional activity for T and Tbx6 provide a potential mechanism for the failure of Tbx6 to functionally replace T and possible competition phenotypes in vivo.

Analysis of Nesprin-2 Interaction with Its Binding Partners and Actin.

Nuclei are connected to the actin cytoskeleton for controlling its position in the cell and for mechanochemical signaling. Nesprin-2G is one of the major outer nuclear membrane proteins that links the nucleus to the actin cytoskeleton. In addition to its paired calponin homology (CH) domains, nesprin-2G interacts with actin filaments by binding the actin-bundling proteins FHOD1 and fascin. We describe methods to measure the interaction of nesprin-2G with actin filaments using an actin co-sedimentation assay and with its binding partner FHOD1 using a GST pull-down method.

Penaeid shrimp brachyury: sequence analysis and expression during gastrulation.

Gastrulation occurs by a variety of morphogenetic movements, often correlated with diverse expression of the T-box transcription factor Brachyury (Bra). Bra may be expressed in ectoderm, mesoderm, or endoderm, but its role in cell fate specification or regulation of gastrulation movements has not been studied in the development of crustaceans. Penaeid shrimp (Decapoda: Dendrobranchiata: Penaeidae) develop by complete cleavage and gastrulation by invagination to a free-swimming nauplius larva. Penaeid gastrulation diverges from other decapods and from insects, occurring early at a low cell number with the formation of a radial invagination. Toward a better understanding of gastrulation movements in penaeid shrimp, bra was identified from newly available penaeid shrimp genomes and transcriptomes of Litopenaeus vannamei, Marsupenaeus japonicus, and Penaeus monodon. Additional bra homologs were obtained from the outgroups Sicyonia ingentis (Decapoda: Dendrobranchiata: Sicyoniidae) and the caridean shrimp Caridina multidentata (Decapoda: Pleocymata). The genes encoded penaeid shrimp Bra proteins of 551-552 amino acids, containing the highly conserved T-box DNA-binding region. The N-terminal Smad1-binding domain, conserved in most animals, was absent in shrimp Bra. The R1 repressor domain was the best conserved of the C-terminal regulatory domains, which were widely divergent compared to other species. The penaeid shrimp bra gene consisted of six exons, with splice sites conserved with other phyla across the animal kingdom. Real-time qPCR and FPKM analysis showed that shrimp bra mRNA was strongly expressed during gastrulation. These findings begin to address the evolution of gastrulation in shrimp at the molecular level.

Helical rotation of the diaphanous-related formin mDia1 generates actin filaments resistant to cofilin.

The complex interplay between actin regulatory proteins facilitates the formation of diverse cellular actin structures. Formin homology proteins (formins) play an essential role in the formation of actin stress fibers and yeast actin cables, to which the major actin depolymerizing factor cofilin barely associates. In vitro, F-actin decorated with cofilin exhibits a marked increase in the filament twist. On the other hand, a mammalian formin mDia1 rotates along the long-pitch actin helix during processive actin elongation (helical rotation). Helical rotation may impose torsional force on F-actin in the opposite direction of the cofilin-induced twisting. Here, we show that helical rotation of mDia1 converts F-actin resistant to cofilin both in vivo and in vitro. F-actin assembled by mDia1 without rotational freedom became more resistant to the severing and binding activities of cofilin than freely rotatable F-actin. Electron micrographic analysis revealed untwisting of the long-pitch helix of F-actin elongating from mDia1 on tethering of both mDia1 and the pointed end side of the filament. In cells, single molecules of mDia1ΔC63, an activated mutant containing N-terminal regulatory domains, showed tethering to cell structures more frequently than autoinhibited wild-type mDia1 and mDia1 devoid of N-terminal domains. Overexpression of mDia1ΔC63 induced the formation of F-actin, which has prolonged lifetime and accelerates dissociation of cofilin. Helical rotation of formins may thus serve as an F-actin stabilizing mechanism by which a barbed end-bound molecule can enhance the stability of a filament over a long range.

Evolution and Classification of the T-Box Transcription Factor Family.

T-box proteins are key developmental transcription factors in Metazoa. Until recently they were thought to be animal specific and many T-box classes were considered bilaterian specific. Recent genome data from both early-branching animals and their closest unicellular relatives have radically changed this scenario. Thus, we now know that T-box genes originated in premetazoans, being present in the genomes of some extant early-branching fungi and unicellular holozoans. Here, we update the evolutionary classification of T-box families and review the evolution of T-box function in early-branching animals (sponges, ctenophores, placozoans, and cnidarians) and nonmodel bilaterians. We show that concomitant with the origin of Metazoa, the T-box family radiated into the major known T-box classes. On the other hand, while functional studies are still missing for many T-box classes, the emerging picture is that T-box genes have key roles in multiple aspects of development and in adult terminal cell-type differentiation in different animal lineages. A paradigmatic example is that of Brachyury, the founding member of the T-box family, for which several studies indicate a widely conserved role in regulating cell motility in different animal lineages and probably even before the advent of animal multicellularity. Overall, we here review the evolutionary history of T-box genes from holozoans to animals and discuss both their functional diversity and conservation.

A network of conserved formins, regulated by the guanine exchange factor EXC-5 and the GTPase CDC-42, modulates tubulogenesis in vivo.

The C. elegans excretory cell (EC) is a powerful model for tubulogenesis, a conserved process that requires precise cytoskeletal regulation. EXC-6, an ortholog of the disease-associated formin INF2, coordinates cell outgrowth and lumen formation during EC tubulogenesis by regulating F-actin at the tip of the growing canal and the dynamics of basolateral microtubules. EXC-6 functions in parallel with EXC-5/FGD, a predicted activator of the Rho GTPase Cdc42. Here, we identify the parallel pathway: EXC-5 functions through CDC-42 to regulate two other formins: INFT-2, another INF2 ortholog, and CYK-1, the sole ortholog of the mammalian diaphanous (mDia) family of formins. We show that INFT-2 promotes F-actin accumulation in the EC, and that CYK-1 inhibits INFT-2 to regulate F-actin levels and EXC-6-promoted outgrowth. As INF2 and mDia physically interact and cross-regulate in cultured cells, our work indicates that a conserved EXC-5-CDC-42 pathway modulates this regulatory interaction and that it is functionally important in vivo during tubulogenesis.

Accelerated actin filament polymerization from microtubule plus ends.

Microtubules (MTs) govern actin network remodeling in a wide range of biological processes, yet the mechanisms underlying this cytoskeletal cross-talk have remained obscure. We used single-molecule fluorescence microscopy to show that the MT plus-end-associated protein CLIP-170 binds tightly to formins to accelerate actin filament elongation. Furthermore, we observed mDia1 dimers and CLIP-170 dimers cotracking growing filament ends for several minutes. CLIP-170-mDia1 complexes promoted actin polymerization ~18 times faster than free-barbed-end growth while simultaneously enhancing protection from capping proteins. We used a MT-actin dynamics co-reconstitution system to observe CLIP-170-mDia1 complexes being recruited to growing MT ends by EB1. The complexes triggered rapid growth of actin filaments that remained attached to the MT surface. These activities of CLIP-170 were required in primary neurons for normal dendritic morphology. Thus, our results reveal a cellular mechanism whereby growing MT plus ends direct rapid actin assembly.

The WH2 Domain and Actin Nucleation: Necessary but Insufficient.

Two types of sequences, proline-rich domains (PRDs) and the WASP-homology 2 (WH2) domain, are found in most actin filament nucleation and elongation factors discovered thus far. PRDs serve as a platform for protein-protein interactions, often mediating the binding of profilin-actin. The WH2 domain is an abundant actin monomer-binding motif comprising ∼17 amino acids. It frequently occurs in tandem repeats, and functions in nucleation by recruiting actin subunits to form the polymerization nucleus. It is found in Spire, Cordon Bleu (Cobl), Leiomodin (Lmod), Arp2/3 complex activators (WASP, WHAMM, WAVE, etc.), the bacterial nucleators VopL/VopF and Sca2, and some formins. Yet, it is argued here that the WH2 domain plays only an auxiliary role in nucleation, always synergizing with other domains or proteins for this activity.

Single-filament kinetic studies provide novel insights into regulation of actin-based motility.

Polarized assembly of actin filaments forms the basis of actin-based motility and is regulated both spatially and temporally. Cells use a variety of mechanisms by which intrinsically slower processes are accelerated, and faster ones decelerated, to match rates observed in vivo. Here we discuss how kinetic studies of individual reactions and cycles that drive actin remodeling have provided a mechanistic and quantitative understanding of such processes. We specifically consider key barbed-end regulators such as capping protein and formins as illustrative examples. We compare and contrast different kinetic approaches, such as the traditional pyrene-polymerization bulk assays, as well as more recently developed single-filament and single-molecule imaging approaches. Recent development of novel biophysical methods for sensing and applying forces will in future allow us to address the very important relationship between mechanical stimulus and kinetics of actin-based motility.

Target-selective photo-degradation of AFP-L3 and selective photo-cytotoxicity against HuH-7 hepatocarcinoma cells using an anthraquinone-PhoSL hybrid.

A purposefully-designed anthraquinone-Pholiota squarrosa lectin (PhoSL) hybrid effectively degraded α-fetoprotein-L3 (AFP-L3) associated with liver cancer. Degradation was achieved under light irradiation in the absence of any additives and under neutral pH conditions. Moreover, the hybrid effectively exhibited selective photo-cytotoxicity against HuH-7 hepatocarcinoma cells upon photo-irradiation.

Structures of CD6 and Its Ligand CD166 Give Insight into Their Interaction.

CD6 is a transmembrane protein with an extracellular region containing three scavenger receptor cysteine rich (SRCR) domains. The membrane proximal domain of CD6 binds the N-terminal immunoglobulin superfamily (IgSF) domain of another cell surface receptor, CD166, which also engages in homophilic interactions. CD6 expression is mainly restricted to T cells, and the interaction between CD6 and CD166 regulates T-cell activation. We have solved the X-ray crystal structures of the three SRCR domains of CD6 and two N-terminal domains of CD166. This first structure of consecutive SRCR domains reveals a nonlinear organization. We characterized the binding sites on CD6 and CD166 and showed that a SNP in CD6 causes glycosylation that hinders the CD6/CD166 interaction. Native mass spectrometry analysis showed that there is competition between the heterophilic and homophilic interactions. These data give insight into how interactions of consecutive SRCR domains are perturbed by SNPs and potential therapeutic reagents.

Identification of human basic fetoprotein as glucose-6-phosphate isomerase by using N- and C-terminal sequence tags and terminal tag database.

Human basic fetoprotein (BFP), found in fetal serum and tissue extracts as well as in extracts of various cancer tissues, has long been known as a marker protein for cancers; however, the primary sequence has not yet been reported. This paper describes the identification of BFP using the N- and C-terminal amino acid sequence tags (Ac-AALTRDPQFQ and QQREARVQ, respectively) clarified by mass spectrometry-based methods, and a terminal tag database (ProteinCarta). In this study, BFP was identified as glucose-6-phosphate isomerase (G6PI_HUMAN).

Optochemical dissection of T-box gene-dependent medial floor plate development.

In addition to their cell-autonomous roles in mesoderm development, the zebrafish T-box transcription factors no tail a (ntla) and spadetail (spt/tbx16) are required for medial floor plate (MFP) formation. Posterior MFP cells are completely absent in zebrafish embryos lacking both Ntla and Spt function, and genetic mosaic analyses have shown that the two T-box genes promote MFP development in a non-cell-autonomous manner. On the basis of these observations, it has been proposed that Ntla/Spt-dependent mesoderm-derived signals are required for the induction of posterior but not anterior MFP cells. To investigate the mechanisms by which Ntla and Spt regulate MFP development, we have used photoactivatable caged morpholinos (cMOs) to silence these T-box genes with spatiotemporal control. We find that posterior MFP formation requires Ntla or Spt activity during early gastrulation, specifically in lateral margin-derived cells that converge toward the midline during epiboly and somitogenesis. Nodal signaling-dependent MFP specification is maintained in the absence of Ntla and Spt function; however, midline cells in ntla;spt morphants exhibit aberrant morphogenetic movements, resulting in their anterior mislocalization. Our findings indicate that Ntla and Spt do not differentially regulate MFP induction along the anterior-posterior axis; rather, the T-box genes act redundantly within margin-derived cells to promote the posterior extension of MFP progenitors.

The acquisition of novel N-glycosylation sites in conserved proteins during human evolution.

BACKGROUND: N-linked protein glycosylation plays an important role in various biological processes, including protein folding and trafficking, and cell adhesion and signaling. The acquisition of a novel N-glycosylation site may have significant effect on protein structure and function, and therefore, on the phenotype. RESULTS: We analyzed the human glycoproteome data set (2,534 N-glycosylation sites in 1,027 proteins) and identified 112 novel N-glycosylation sites in 91 proteins that arose in the human lineage since the last common ancestor of Euarchonta (primates and treeshrews). Three of them, Asn-196 in adipocyte plasma membrane-associated protein (APMAP), Asn-91 in cluster of differentiation 166 (CD166/ALCAM), and Asn-76 in thyroglobulin, are human-specific. Molecular evolutionary analysis suggested that these sites were under positive selection during human evolution. Notably, the Asn-76 of thyroglobulin might be involved in the increased production of thyroid hormones in humans, especially thyroxine (T4), because the removal of the glycan moiety from this site was reported to result in a significant decrease in T4 production. CONCLUSIONS: We propose that the novel N-glycosylation sites described in this study may be useful candidates for functional analyses to identify innovative genetic modifications for beneficial phenotypes acquired in the human lineage.

Unique features of apicoplast DNA gyrases from Toxoplasma gondii and Plasmodium falciparum.

BACKGROUND: DNA gyrase, an enzyme once thought to be unique to bacteria, is also found in some eukaryotic plastids including the apicoplast of Apicomplexa such as Plasmodium falciparum and Toxoplasma gondii which are important disease-causing organisms. DNA gyrase is an excellent target for antibacterial drugs, yet such antibacterials seem ineffective against Apicomplexa. Characterisation of the apicoplast gyrases would be a useful step towards understanding why this should be so. While purification of active apicoplast gyrase has proved impossible to date, in silico analyses have allowed us to discover differences in the apicoplast proteins. The resulting predicted structural and functional differences will be a first step towards development of apicoplast-gyrase specific inhibitors. RESULTS: We have carried out sequence analysis and structural predictions of the enzymes from the two species and find that P. falciparum gyrase lacks a GyrA box, but T. gondii may retain one. All proteins contained signal/transport peptides for localization to the apicoplast but T. gondii Gyrase B protein lacks the expected hydrophobic region. The most significant difference is in the GyrA C-terminal domain: While the cores of the proteins, including DNA binding and cleavage regions are essentially unchanged, both apicoplast gyrase A proteins have C-terminal domains that are significantly larger than bacterial counterparts and are predicted to have different structures. CONCLUSION: The apicoplast gyrases differ significantly from bacterial gyrases while retaining similar core domains. T. gondii Gyrase B may have an unusual or inefficient mechanism of localisation to the apicoplast. P.falciparum gyrase, lacks a GyrA box and is therefore likely to be inefficient in DNA supercoiling. The C-terminal domains of both apicoplast Gyrase A proteins diverge significantly from the bacterial proteins. We predict that an additional structural element is present in the C-terminal domain of both apicoplast Gyrase A proteins, including the possibility of a ß-pinwheel with a non-canonical number of blades. These differences undoubtedly will affect the DNA supercoiling mechanism and have perhaps evolved to compensate for the lack of Topoisomerase IV in the apicoplast. These data will be useful first step towards further characterisation and development of inhibitors for apicoplast gyrases.

Formins as effector proteins of Rho GTPases.

Formin proteins were recognized as effectors of Rho GTPases some 15 years ago. They contribute to different cellular actin cytoskeleton structures by their ability to polymerize straight actin filaments at the barbed end. While not all formins necessarily interact with Rho GTPases, a subgroup of mammalian formins, termed Diaphanous-related formins or DRFs, were shown to be activated by small GTPases of the Rho superfamily. DRFs are autoinhibited in the resting state by an N- to C-terminal interaction that renders the central actin polymerization domain inactive. Upon the interaction with a GTP-bound Rho, Rac, or Cdc42 GTPase, the C-terminal autoregulation domain is displaced from its N-terminal recognition site and the formin becomes active to polymerize actin filaments. In this review we discuss the current knowledge on the structure, activation, and function of formin-GTPase interactions for the mammalian formin families Dia, Daam, FMNL, and FHOD. We describe both direct and indirect interactions of formins with GTPases, which lead to formin activation and cytoskeletal rearrangements. The multifaceted function of formins as effector proteins of Rho GTPases thus reflects the diversity of the actin cytoskeleton in cells.

Differential pharmacological activity of JN403 between α7 and muscle nicotinic acetylcholine receptors.

The differential action of the novel agonist JN403 at neuronal α7 and muscle nicotinic receptors (AChRs) was explored by using a combination of functional and structural approaches. Single-channel recordings reveal that JN403 is a potent agonist of α7 but a very low-efficacy agonist of muscle AChRs. JN403 elicits detectable openings of α7 and muscle AChRs at concentrations ~1000-fold lower and ~20-fold higher, respectively, than that for ACh. Single-channel activity elicited by JN403 is very similar to that elicited by ACh in α7 but profoundly different in muscle AChRs, where openings are brief and infrequent and do not appear in clusters at any concentration. JN403 elicits single-channel activity of muscle AChRs lacking the ε subunit, with opening events being more frequent and prolonged than those of wild-type AChRs. This finding is in line with the molecular docking studies predicting that JN403 may form a hydrogen bond required for potent activation at the α-δ but not at the α-ε binding site. JN403 does not elicit detectable Ca²âº influx in muscle AChRs but inhibits (±)-epibatidine-elicited influx mainly by a noncompetitive mechanism. Such inhibition is compatible with single-channel recordings revealing that JN403 produces open-channel blockade and early termination of ACh-elicited clusters, and it is therefore also a potent desensitizing enhancer of muscle AChRs. The latter mechanism is supported by the JN403-induced increase in the level of binding of [³H]cytisine and [³H]TCP to resting AChRs. Elucidation of the differences in activity of JN403 between neuronal α7 and muscle AChRs provides further insights into mechanisms underlying selectivity for α7 AChRs.

Multiple mutant T alleles cause haploinsufficiency of Brachyury and short tails in Manx cats.

Most mammals possess a tail, humans and the Great Apes being notable exceptions. One approach to understanding the mechanisms and evolutionary forces influencing development of a tail is to identify the genetic factors that influence extreme tail length variation within a species. In mice, the Tailless locus has proven to be complex, with evidence of multiple different genes and mutations with pleiotropic effects on tail length, fertility, embryogenesis, male transmission ratio, and meiotic recombination. Five cat breeds have abnormal tail length phenotypes: the American Bobtail, the Manx, the Pixie-Bob, the Kurilian Bobtail, and the Japanese Bobtail. We sequenced the T gene in several independent lineages of Manx cats from both the US and the Isle of Man and identified three 1-bp deletions and one duplication/deletion, each predicted to cause a frameshift that leads to premature termination and truncation of the carboxy terminal end of the Brachyury protein. Ninety-five percent of Manx cats with short-tail phenotypes were heterozygous for T mutations, mutant alleles appeared to be largely lineage-specific, and a maximum LOD score of 6.21 with T was obtained at a recombination fraction (Θ) of 0.00. One mutant T allele was shared with American Bobtails and Pixie-Bobs; both breeds developed more recently in the US. The ability of mutant Brachyury protein to activate transcription of a downstream target was substantially lower than wild-type protein. Collectively, these results suggest that haploinsufficiency of Brachyury is one mechanism underlying variable tail length in domesticated cats.

Interaction of formin FH2 with skeletal muscle actin. EPR and DSC studies.

Formins are highly conserved proteins that are essential in the formation and regulation of the actin cytoskeleton. The formin homology 2 (FH2) domain is responsible for actin binding and acts as an important nucleating factor in eukaryotic cells. In this work EPR and DSC were used to investigate the properties of the mDia1-FH2 formin fragment and its interaction with actin. MDia1-FH2 was labeled with a maleimide spin probe (MSL). EPR results suggested that the MSL was attached to a single SH group in the FH2. In DSC and temperature-dependent EPR experiments we observed that mDia1-FH2 has a flexible structure and observed a major temperature-induced conformational change at 41 °C. The results also confirmed the previous observation obtained by fluorescence methods that formin binding can destabilize the structure of actin filaments. In the EPR experiments the intermolecular connection between the monomers of formin dimers proved to be flexible. Considering the complex molecular mechanisms underlying the cellular roles of formins this internal flexibility of the dimers is probably important for manifestation of their biological functions.