The role of Y14 in regulating mRNA decay and P-body formation is described. Y14 interacts with Dcp2 and inhibits mRNA decapping. Moreover, Y14 prevents mRNA degradation and is essential for P-body formation. Y14 may function independent of the EJC to counteract mRNA decay in mRNA metabolism.

The exon-junction complex (EJC) deposited on a newly spliced mRNA plays an important role in subsequent mRNA metabolic events. Here we show that an EJC core heterodimer, Y14/Magoh, specifically associates with mRNA-degradation factors, including the mRNA-decapping complex and exoribonucleases, whereas another core factor, eIF4AIII/MLN51, does not. We also demonstrate that Y14 interacts directly with the decapping factor Dcp2 and the 5′ cap structure of mRNAs via different but overlapping domains and that Y14 inhibits the mRNA-decapping activity of Dcp2 in vitro. Accordingly, overexpression of Y14 prolongs the half-life of a reporter mRNA. Therefore Y14 may function independently of the EJC in preventing mRNA decapping and decay. Furthermore, we observe that depletion of Y14 disrupts the formation of processing bodies, whereas overexpression of a phosphomimetic Y14 considerably increases the number of processing bodies, perhaps by sequestering the mRNA-degradation factors. In conclusion, this report provides unprecedented evidence for a role of Y14 in regulating mRNA degradation and processing body formation and reinforces the influence of phosphorylation of Y14 on its activity in postsplicing mRNA metabolism.

Endoplasmic reticulum (ER) stress induces an mRNA decay pathway termed regulated Ire-dependent decay (RIDD). This study shows that in Drosophila cells, ER association is sufficient for targeting mRNAs to RIDD and most membrane-associated mRNAs are degraded more rapidly during ER stress. It is also reported that a small number of mRNAs are specifically protected from this default pathway.

Ire1 is an endoplasmic reticulum (ER) transmembrane protein that senses disturbances in protein folding homeostasis and contributes to a multifaceted response to stress. The nuclease activity of Ire1, in addition to splicing the mRNA encoding the transcription factor Xbp1, mediates mRNA degradation in response to ER stress through a pathway termed regulated Ire1-dependent decay (RIDD). We previously showed that ER targeting of substrates is necessary for RIDD; in this paper, we show that ER localization is also sufficient to induce decay in a normally unaffected mRNA. Using microarrays, we also measured relative mRNA degradation in the presence and absence of ER stress in Drosophila S2 cells, and determined mRNA membrane association using detergent fractionation. The vast majority of mRNAs that were strongly associated with the ER were degraded faster during ER stress in an Ire1-dependent manner, suggesting that RIDD is the default pathway for ER-localized mRNAs during stress. We also show that the mRNA encoding plexin A remains highly polysome associated during stress and escapes degradation by RIDD, and that its 5′ untranslated region can protect a strong RIDD target from degradation. These results suggest that while translation is generally attenuated during ER stress, continued translation of certain messages can protect them from degradation by RIDD.

Interaction of vimentin filaments (vIFs) and force-bearing adhesions is essential for endoplasm spreading. For adhesions to be connected to a contractile network involved in endoplasm spreading, vIFs are needed. Thus endoplasm spreading and microtubule stabilization in the periphery require a multicomponent actin network anchored at adhesions.

For cells to develop long-range forces and carry materials to the periphery, the microtubule and organelle-rich region at the center of the cell—the endoplasm—needs to extend to near the cell edge. Depletion of the actin cross-linking protein filamin A (FlnA) causes a collapse of the endoplasm into a sphere around the nucleus of fibroblasts and disruption of matrix adhesions, indicating that FlnA is involved in endoplasmic spreading and adhesion growth. Here, we report that treatment with the calpain inhibitor N-[N-(N-acetyl-l-leucyl)-l-leucyl]-l-norleucine (ALLN) restores endoplasmic spreading as well as focal adhesion (FA) growth on fibronectin-coated surfaces in a Fln-depleted background. Addback of calpain-uncleavable talin, not full-length talin, achieves a similar effect in Fln-depleted cells and indicates a crucial role for talin in endoplasmic spreading. Because FA maturation involves the vimentin intermediate filament (vIF) network, we also examined the role of vIFs in endoplasmic spreading. Wild-type cells expressing a vimentin variant incapable of polymerization exhibit deficient endoplasmic spreading as well as defects in FA growth. ALLN treatment restores FA growth despite the lack of vIFs but does not restore endoplasmic spreading, implying that vIFs are essential for endoplasm spreading. Consistent with that hypothesis, vIFs are always displaced from adhesions when the endoplasm does not spread. In Fln-depleted cells, vIFs extend beyond adhesions, nearly to the cell edge. Finally, inhibiting myosin II–mediated contraction blocks endoplasmic spreading and adhesion growth. Thus we propose a model in which myosin II–mediated forces and coalescence of vIFs at mature FAs are required for endoplasmic spreading.

Dual-color total internal reflection fluorescence microscopy revealed that the N-terminal half of Srv2 (N-Srv2) directly catalyzes severing of cofilin-decorated actin filaments. N-Srv2 formed novel six-bladed structures resembling ninja throwing stars (shurikens), and N-Srv2 activities were critical for actin organization in vivo and were lethal in combination with Aip1.

Actin filament severing is critical for the dynamic turnover of cellular actin networks. Cofilin severs filaments, but additional factors may be required to increase severing efficiency in vivo. Srv2/cyclase-associated protein (CAP) is a widely expressed protein with a role in binding and recycling actin monomers ascribed to domains in its C-terminus (C-Srv2). In this paper, we report a new biochemical and cellular function for Srv2/CAP in directly catalyzing cofilin-mediated severing of filaments. This function is mediated by its N-terminal half (N-Srv2), and is physically and genetically separable from C-Srv2 activities. Using dual-color total internal reflection fluorescence microscopy, we determined that N-Srv2 stimulates filament disassembly by increasing the frequency of cofilin-mediated severing without affecting cofilin binding to filaments. Structural analysis shows that N-Srv2 forms novel hexameric star-shaped structures, and disrupting oligomerization impairs N-Srv2 activities and in vivo function. Further, genetic analysis shows that the combined activities of N-Srv2 and Aip1 are essential in vivo. These observations define a novel mechanism by which the combined activities of cofilin and Srv2/CAP lead to enhanced filament severing and support an emerging view that actin disassembly is controlled not by cofilin alone, but by a more complex set of factors working in concert.

This article reports that TLR9 signaling can reduce intrinsic microRNA-7 (miR-7) expression in human lung cancer cells and that overexpression of miR-7 can significantly inhibit TLR9 signaling–enhanced growth and metastatic potential of lung cancer cells in vitro and in vivo.

Recent evidence shows that microRNAs (miRNAs) contribute to the biological effects of Toll-like receptor (TLR) signaling on various cells. Our previous data showed that TLR9 signaling could enhance the growth and metastatic potential of human lung cancer cells. However, the potential role of miRNAs in the effects of TLR9 signaling on tumor biology remains unknown. In this paper, we first report that TLR9 signaling could reduce intrinsic miR-7 expression in human lung cancer cells. Furthermore, overexpression of miR-7 can significantly inhibit TLR9 signaling–enhanced growth and metastatic potential of lung cancer cells in vitro and in vivo. Notably, we identify phosphoinositide-3-kinase, regulatory subunit 3 (PIK3R3) as a novel target molecule of miR-7 in lung cancer cells by Western blotting and luciferase report assay. Further study shows that miR-7 inhibits the effects of TLR9 signaling on lung cancer cells through regulation of the PIK3R3/Akt pathway. These data suggest that miR-7 could act as a fine-tuner in regulating the biological effects of TLR9 signaling on human lung cancer cells, which might be helpful to the understanding of the potential role of miRNAs in TLR signaling effects on tumor biology.

Basal bodies act as anchorage sites for cilia and resist forces generated by ciliary beating. Bld10/Cep135 is a basal body assembly factor. A novel role is found for Bld10 in stabilizing basal bodies to resist cilia-generated forces. Bld10 achieves this by stabilizing the A- and C-tubules of the basal body triplet microtubule blades.

Basal bodies nucleate, anchor, and organize cilia. As the anchor for motile cilia, basal bodies must be resistant to the forces directed toward the cell as a consequence of ciliary beating. The molecules and generalized mechanisms that contribute to the maintenance of basal bodies remain to be discovered. Bld10/Cep135 is a basal body outer cartwheel domain protein that has established roles in the assembly of nascent basal bodies. We find that Bld10 protein first incorporates stably at basal bodies early during new assembly. Bld10 protein continues to accumulate at basal bodies after assembly, and we hypothesize that the full complement of Bld10 is required to stabilize basal bodies. We identify a novel mechanism for Bld10/Cep135 in basal body maintenance so that basal bodies can withstand the forces produced by motile cilia. Bld10 stabilizes basal bodies by promoting the stability of the A- and C-tubules of the basal body triplet microtubules and by properly positioning the triplet microtubule blades. The forces generated by ciliary beating promote basal body disassembly in bld10Δ cells. Thus Bld10/Cep135 acts to maintain the structural integrity of basal bodies against the forces of ciliary beating in addition to its separable role in basal body assembly.

The advent of ultra–high-throughput sequencing has led to the discovery of a large group of small, noncoding RNAs that are not microRNAs. The functional relevance of microRNAs has been well established over the last decade. In this Perspective, we focus on the non-micro-short RNAs that comprise a variety of functional classes and range from 16–40 nucleotides in size. We will highlight how some of these non-micro-short RNAs were discovered, as well as their biogenesis, potential mechanisms of action, and role in diverse biological processes, development, and disease. Finally, we will describe what must be done to further our understanding of these enigmatic molecules.

Misfolded protein studies of endoplasmic reticulum–associated degradation have relied on overexpressed model substrates, and the identities of endogenous substrates are largely unknown. This study used a proteomic approach to identify unassembled CD147 as an endogenous substrate of the OS-9/SEL1L/Hrd1 ubiquitin ligase.

Degradation of folding- or assembly-defective proteins by the endoplasmic reticulum–associated degradation (ERAD) ubiquitin ligase, Hrd1, is facilitated by a process that involves recognition of demannosylated N-glycans by the lectin OS-9/XTP3-B via the adaptor protein SEL1L. Most of our knowledge of the machinery that commits proteins to this fate in metazoans comes from studies of overexpressed mutant proteins in heterologous cells. In this study, we used mass spectrometry to identify core-glycoslyated CD147 (CD147(CG)) as an endogenous substrate of the ERAD system that accumulates in a complex with OS-9 following SEL1L depletion. CD147 is an obligatory assembly factor for monocarboxylate transporters. The majority of newly synthesized endogenous CD147(CG) was degraded by the proteasome in a Hrd1-dependent manner. CD147(CG) turnover was blocked by kifunensine, and interaction of OS-9 and XTP3-B with CD147(CG) was inhibited by mutations to conserved residues in their lectin domains. These data establish unassembled CD147(CG) as an endogenous, constitutive ERAD substrate of the OS-9/SEL1L/Hrd1 pathway.

The transition of proteins targeted for autophagic degradation from the soluble to the insoluble phase is regulated in an ATG1-independent mechanism by TORC1. This process is likely a critical mechanism for maintaining protein homeostasis when challenged with proteomic stress.

Accumulation of insoluble protein in cells is associated with aging and aging-related diseases; however, the roles of insoluble protein in these processes are uncertain. The nature and impact of changes to protein solubility during normal aging are less well understood. Using quantitative mass spectrometry, we identify 480 proteins that become insoluble during postmitotic aging in Saccharomyces cerevisiae and show that this ensemble of insoluble proteins is similar to those that accumulate in aging nematodes. SDS-insoluble protein is present exclusively in a nonquiescent subpopulation of postmitotic cells, indicating an asymmetrical distribution of this protein. In addition, we show that nitrogen starvation of young cells is sufficient to cause accumulation of a similar group of insoluble proteins. Although many of the insoluble proteins identified are known to be autophagic substrates, induction of macroautophagy is not required for insoluble protein formation. However, genetic or chemical inhibition of the Tor1 kinase is sufficient to promote accumulation of insoluble protein. We conclude that target of rapamycin complex 1 regulates accumulation of insoluble proteins via mechanisms acting upstream of macroautophagy. Our data indicate that the accumulation of proteins in an SDS-insoluble state in postmitotic cells represents a novel autophagic cargo preparation process that is regulated by the Tor1 kinase.

Arachidonic acid (AA) is remarkably enriched in phosphatidylinositol (PI). Studies using knockout mice of lysophosphatidylinositol acyltransferase 1, which selectively incorporates AA into PI, reveal that AA-containing PI plays a crucial role in cortical lamination and neuronal migration during brain development.

Dietary arachidonic acid (AA) has roles in growth, neuronal development, and cognitive function in infants. AA is remarkably enriched in phosphatidylinositol (PI), an important constituent of biological membranes in mammals; however, the physiological significance of AA-containing PI remains unknown. In an RNA interference–based genetic screen using Caenorhabditis elegans, we recently cloned mboa-7 as an acyltransferase that selectively incorporates AA into PI. Here we show that lysophosphatidylinositol acyltransferase 1 (LPIAT1, also known as MBOAT7), the closest mammalian homologue, plays a crucial role in brain development in mice. Lpiat1−/− mice show almost no LPIAT activity with arachidonoyl-CoA as an acyl donor and show reduced AA contents in PI and PI phosphates. Lpiat1−/− mice die within a month and show atrophy of the cerebral cortex and hippocampus. Immunohistochemical analysis reveals disordered cortical lamination and delayed neuronal migration in the cortex of E18.5 Lpiat1−/− mice. LPIAT1 deficiency also causes disordered neuronal processes in the cortex and reduced neurite outgrowth in vitro. Taken together, these results demonstrate that AA-containing PI/PI phosphates play an important role in normal cortical lamination during brain development in mice.

Semliki Forest virus nsP3 sequesters G3BP to inhibit stress granule formation on viral mRNAs. Furthermore, the efficient translation of viral mRNAs containing a translation enhancer element assists disruption of SGs in infected cells. This work thus describes a novel mechanism for SG disruption.

Dynamic, mRNA-containing stress granules (SGs) form in the cytoplasm of cells under environmental stresses, including viral infection. Many viruses appear to employ mechanisms to disrupt the formation of SGs on their mRNAs, suggesting that they represent a cellular defense against infection. Here, we report that early in Semliki Forest virus infection, the C-terminal domain of the viral nonstructural protein 3 (nsP3) forms a complex with Ras-GAP SH3-domain–binding protein (G3BP) and sequesters it into viral RNA replication complexes in a manner that inhibits the formation of SGs on viral mRNAs. A viral mutant carrying a C-terminal truncation of nsP3 induces more persistent SGs and is attenuated for propagation in cell culture. Of importance, we also show that the efficient translation of viral mRNAs containing a translation enhancer sequence also contributes to the disassembly of SGs in infected cells. Furthermore, we show that the nsP3/G3BP interaction also blocks SGs induced by other stresses than virus infection. This is one of few described viral mechanisms for SG disruption and underlines the role of SGs in antiviral defense.

Proper coupling of centrosomes to the nuclear surface at prophase is essential for fidelity of mitotic events. A pool of dynein motors anchored to the nuclear surface mediates this step. The protein Asunder is required in human cultured cells for dynein localization and tethering of centrosomes to the nucleus at mitotic entry.

Recruitment of dynein motors to the nuclear surface is an essential step for nucleus–centrosome coupling in prophase. In cultured human cells, this dynein pool is anchored to nuclear pore complexes through RanBP2–Bicaudal D2 (BICD2) and Nup133– centromere protein F (CENP-F) networks. We previously reported that the asunder (asun) gene is required in Drosophila spermatocytes for perinuclear dynein localization and nucleus–centrosome coupling at G2/M of male meiosis. We show here that male germline expression of mammalian Asunder (ASUN) protein rescues asun flies, demonstrating evolutionary conservation of function. In cultured human cells, we find that ASUN down-regulation causes reduction of perinuclear dynein in prophase of mitosis. Additional defects after loss of ASUN include nucleus–centrosome uncoupling, abnormal spindles, and multinucleation. Coimmunoprecipitation and overlapping localization patterns of ASUN and lissencephaly 1 (LIS1), a dynein adaptor, suggest that ASUN interacts with dynein in the cytoplasm via LIS1. Our data indicate that ASUN controls dynein localization via a mechanism distinct from that of either BICD2 or CENP-F. We present a model in which ASUN promotes perinuclear enrichment of dynein at G2/M that facilitates BICD2- and CENP-F-mediated anchoring of dynein to nuclear pore complexes.

Zebrafish mutants in Fn1 and Itga5 possess cataracts, characterized by defects in fiber adhesion, elongation, and packing. Analysis of the intracellular mediators of integrin α5/Fn1 activity FAK and ILK reveals that FAK, but not ILK, is also required for lens fiber morphogenesis and that it acts cell autonomously during lens fiber morphogenesis.

Lens fiber formation and morphogenesis requires a precise orchestration of cell– extracellular matrix (ECM) and cell–cell adhesive changes in order for a lens epithelial cell to adopt a lens fiber fate, morphology, and migratory ability. The cell–ECM interactions that mediate these processes are largely unknown, and here we demonstrate that fibronectin1 (Fn1), an ECM component, and integrin α5, its cellular binding partner, are required in the zebrafish lens for fiber morphogenesis. Mutations compromising either of these proteins lead to cataracts, characterized by defects in fiber adhesion, elongation, and packing. Loss of integrin α5/Fn1 does not affect the fate or viability of lens epithelial cells, nor does it affect the expression of differentiation markers expressed in lens fibers, although nucleus degradation is compromised. Analysis of the intracellular mediators of integrin α5/Fn1 activity focal adhesion kinase (FAK) and integrin-linked kinase (ILK) reveals that FAK, but not ILK, is also required for lens fiber morphogenesis. These results support a model in which lens fiber cells use integrin α5 to migrate along a Fn-containing substrate on the apical side of the lens epithelium and on the posterior lens capsule, likely activating an intracellular signaling cascade mediated by FAK in order to orchestrate the cytoskeletal changes in lens fibers that facilitate elongation, migration, and compaction.

In three-dimensional matrices, tumor cells can move with either an elongated/mesenchymal or a round/amoeboid morphology. This study identifies FilGAP, a Rac GTPase-activating protein (GAP), as a mediator of Rho/Rho-associated protein kinase–dependent amoeboid movement of carcinoma cells. FilGAP may contribute to tumor invasion and metastasis by controlling the amoeboid mode of movement.

Tumor cells exhibit two interconvertible modes of cell motility referred to as mesenchymal and amoeboid migration. Mesenchymal mode is characterized by elongated morphology that requires high GTPase Rac activation, whereas amoeboid mode is dependent on actomyosin contractility induced by Rho/Rho-associated protein kinase (ROCK) signaling. While elongated morphology is driven by Rac-induced protrusion at the leading edge, how Rho/ROCK signaling controls amoeboid movement is not well understood. We identified FilGAP, a Rac GTPase-activating protein (GAP), as a mediator of Rho/ROCK-dependent amoeboid movement of carcinoma cells. We show that depletion of endogenous FilGAP in carcinoma cells induced highly elongated mesenchymal morphology. Conversely, forced expression of FilGAP induced a round/amoeboid morphology that requires Rho/ROCK-dependent phosphorylation of FilGAP. Moreover, depletion of FilGAP impaired breast cancer cell invasion through extracellular matrices and reduced tumor cell extravasation in vivo. Thus phosphorylation of FilGAP by ROCK appears to promote amoeboid morphology of carcinoma cells, and FilGAP contributes to tumor invasion.

Various agonists trigger β2-integrin activation in neutrophils, yet the mechanisms that regulate β2-integrin inside-out signaling remain obscure. Radil, a novel Rap downstream effector, is an important adapter in the pathway that links G protein–coupled chemoattractant receptors to adhesion complexes during neutrophil chemotaxis.

Integrin activation is required to facilitate multiple adhesion-dependent functions of neutrophils, such as chemotaxis, which is critical for inflammatory responses to injury and pathogens. However, little is known about the mechanisms that mediate integrin activation in neutrophils. We show that Radil, a novel Rap1 effector, regulates β1- and β2-integrin activation and controls neutrophil chemotaxis. On activation and chemotactic migration of neutrophils, Radil quickly translocates from the cytoplasm to the plasma membrane in a Rap1a-GTP–dependent manner. Cells overexpressing Radil show a substantial increase in cell adhesion, as well as in integrin/focal adhesion kinase (FAK) activation, and exhibit an elongated morphology, with severe tail retraction defects. This phenotype is effectively rescued by treatment with either β2-integrin inhibitory antibodies or FAK inhibitors. Conversely, knockdown of Radil causes severe inhibition of cell adhesion, β2-integrin activation, and chemotaxis. Furthermore, we found that inhibition of Rap activity by RapGAP coexpression inhibits Radil-mediated integrin and FAK activation, decreases cell adhesion, and abrogates the long-tail phenotype of Radil cells. Overall, these studies establish that Radil regulates neutrophil adhesion and motility by linking Rap1 to β2-integrin activation.

Centrins are a family of small calcium-binding proteins found at basal bodies. The family can be separated into two groups based on sequence homology to the human centrins. The model organism Tetrahymena was used to show that the two centrin groups have similar but distinct functions at basal bodies.

Centrins are a ubiquitous family of small Ca2+-binding proteins found at basal bodies that are placed into two groups based on sequence similarity to the human centrins 2 and 3. Analyses of basal body composition in different species suggest that they contain a centrin isoform from each group. We used the ciliate protist Tetrahymena thermophila to gain a better understanding of the functions of the two centrin groups and to determine their potential redundancy. We have previously shown that the Tetrahymena centrin 1 (Cen1), a human centrin 2 homologue, is required for proper basal body function. In this paper, we show that the Tetrahymena centrin 2 (Cen2), a human centrin 3 homologue, has functions similar to Cen1 in basal body orientation, maintenance, and separation. The two are, however, not redundant. A further examination of human centrin 3 homologues shows that they function in a manner distinct from human centrin 2 homologues. Our data suggest that basal bodies require a centrin from both groups in order to function correctly.

Lysine methyltransferase G9a interacts with the transcription factor Sharp-1 and enhances transcriptional repression of muscle promoters. RNAi-mediated reduction of G9a expression or inhibition of its activity rescues myogenic differentiation in Sharp-1–overexpressing cells.

Sharp-1, a basic helix-loop-helix transcription factor, is a potent repressor of skeletal muscle differentiation and is dysregulated in muscle pathologies. However, the mechanisms by which it inhibits myogenesis are not fully understood. Here we show that G9a, a lysine methyltransferase, is involved in Sharp-1–mediated inhibition of muscle differentiation. We demonstrate that G9a directly interacts with Sharp-1 and enhances its ability to transcriptionally repress the myogenin promoter. Concomitant with a differentiation block, G9a-dependent histone H3 lysine 9 dimethylation (H3K9me2) and MyoD methylation are apparent upon Sharp-1 overexpression in muscle cells. RNA interference–mediated reduction of G9a or pharmacological inhibition of its activity erases these repressive marks and rescues the differentiation defect imposed by Sharp-1. Our findings provide new insights into Sharp-1–dependent regulation of myogenesis and identify epigenetic mechanisms that could be targeted in myopathies characterized by elevated Sharp-1 levels.

The N-terminus of the kinesin-13 family (Kif2a, Kif2b, Kif2c) is the primary localization determinant. However, the C-terminus of Kif2b associates with Cep170 and Cep170R to create targeting specificity. Cep170 has microtubule-binding properties in vitro and provides a second microtubule-binding site to Kif2b to target it to the spindle.

Microtubule dynamics are essential throughout mitosis to ensure correct chromosome segregation. Microtubule depolymerization is controlled in part by microtubule depolymerases, including the kinesin-13 family of proteins. In humans, there are three closely related kinesin-13 isoforms (Kif2a, Kif2b, and Kif2c/MCAK), which are highly conserved in their primary sequences but display distinct localization and nonoverlapping functions. Here we demonstrate that the N-terminus is a primary determinant of kinesin-13 localization. However, we also find that differences in the C-terminus alter the properties of kinesin-13, in part by facilitating unique protein–protein interactions. We identify the spindle-localized proteins Cep170 and Cep170R (KIAA0284) as specifically associating with Kif2b. Cep170 binds to microtubules in vitro and provides Kif2b with a second microtubule-binding site to target it to the spindle. Thus the intrinsic properties of kinesin-13s and extrinsic factors such as their associated proteins result in the diversity and specificity within the kinesin-13 depolymerase family.

Cosedimentation assays and fluorescence microscopy are used to show that microtubules (MTs) induce Tau filament formation under normal MT-binding assay conditions. This filament formation explains the striking inconsistencies in previously reported Tau–MT affinities and may provide a new model for Tau filament formation in Alzheimer's disease.

Tau is a neuronal protein that stabilizes the microtubule (MT) network, but it also forms filaments associated with Alzheimer's disease. Understanding Tau–MT and Tau–Tau interactions would help to establish Tau function in health and disease. For many years, literature reports on Tau–MT binding behavior and affinity have remained surprisingly contradictory (e.g., 10-fold variation in Tau–MT affinity). Tau–Tau interactions have also been investigated, but whether MTs might affect Tau filament formation is unknown. We have addressed these issues through binding assays and microscopy. We assessed Tau–MT interactions via cosedimentation and found that the measured affinity of Tau varies greatly, depending on the experimental design and the protein concentrations used. To investigate this dependence, we used fluorescence microscopy to examine Tau–MT binding. Strikingly, we found that Taxol-stabilized MTs promote Tau filament formation without characterized Tau-filament inducers. We propose that these novel Tau filaments account for the incongruence in Tau–MT affinity measurements. Moreover, electron microscopy reveals that these filaments appear similar to the heparin-induced Alzheimer's model. These observations suggest that the MT-induced Tau filaments provide a new model for Alzheimer's studies and that MTs might play a role in the formation of Alzheimer's-associated neurofibrillary tangles.

RhoD has a role in actin dynamics that is distinct from RhoA, Rac1, and Cdc42. Data presented here indicate that RhoD binds the actin nucleation–promoting factor WASp homologue associated with actin Golgi membranes and microtubules (WHAMM) and the related filamin A–binding protein FILIP1. WHAMM acts downstream of RhoD, and both proteins coordinate vital cellular processes, such as actin dynamics, cell attachment, and cell migration.

The Rho GTPases have mainly been studied in association with their roles in the regulation of actin filament organization. These studies have shown that the Rho GTPases are essential for basic cellular processes, such as cell migration, contraction, and division. In this paper, we report that RhoD has a role in the organization of actin dynamics that is distinct from the roles of the better-studied Rho members Cdc42, RhoA, and Rac1. We found that RhoD binds the actin nucleation–promoting factor WASp homologue associated with actin Golgi membranes and microtubules (WHAMM), as well as the related filamin A–binding protein FILIP1. Of these two RhoD-binding proteins, WHAMM was found to bind to the Arp2/3 complex, while FILIP1 bound filamin A. WHAMM was found to act downstream of RhoD in regulating cytoskeletal dynamics. In addition, cells treated with small interfering RNAs for RhoD and WHAMM showed increased cell attachment and decreased cell migration. These major effects on cytoskeletal dynamics indicate that RhoD and its effectors control vital cytoskeleton-driven cellular processes. In agreement with this notion, our data suggest that RhoD coordinates Arp2/3-dependent and FLNa-dependent mechanisms to control the actin filament system, cell adhesion, and cell migration.

5-HT promotes neurite growth via IP3-dependent Ca2+ release in neuronal growth cones. Outgrowth depends on increased rates of actin array treadmilling mediated by Ca2+-calcineurin–dependent cofilin activation. This mode of growth contrasts with substrate-dependent responses, for which retrograde actin flow and advance rates have been inversely correlated.

Neurite outgrowth in response to soluble growth factors often involves changes in intracellular Ca2+; however, mechanistic roles for Ca2+ in controlling the underlying dynamic cytoskeletal processes have remained enigmatic. Bag cell neurons exposed to serotonin (5-hydroxytryptamine [5-HT]) respond with a threefold increase in neurite outgrowth rates. Outgrowth depends on phospholipase C (PLC) → inositol trisphosphate → Ca2+ → calcineurin signaling and is accompanied by increased rates of retrograde actin network flow in the growth cone P domain. Calcineurin inhibitors had no effect on Ca2+ release or basal levels of retrograde actin flow; however, they completely suppressed 5-HT–dependent outgrowth and F-actin flow acceleration. 5-HT treatments were accompanied by calcineurin-dependent increases in cofilin activity in the growth cone P domain. 5-HT effects were mimicked by direct activation of PLC, suggesting that increased actin network treadmilling may be a widespread mechanism for promoting neurite outgrowth in response to neurotrophic factors.

Using macrophages overexpressing or reducing SNAP-23, this study shows that SNAP-23 is implicated in phagosome formation and maturation, presumably by mediating SNARE-based membrane traffic. Indeed, a conformational change in SNAP-23 structure based on FRET signal is observed on the phagosome membrane of cells overexpressing the lysosomal SNARE VAMP7.

Synaptosomal associated protein of 23 kDa (SNAP-23), a plasma membrane–localized soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE), has been implicated in phagocytosis by macrophages. For elucidation of its precise role in this process, a macrophage line overexpressing monomeric Venus–tagged SNAP-23 was established. These cells showed enhanced Fc receptor–mediated phagocytosis. Detailed analyses of each process of phagocytosis revealed a marked increase in the production of reactive oxygen species within phagosomes. Also, enhanced accumulation of a lysotropic dye, as well as augmented quenching of a pH-sensitive fluorophore were observed. Analyses of isolated phagosomes indicated the critical role of SNAP-23 in the functional recruitment of the NADPH oxidase complex and vacuolar-type H+-ATPase to phagosomes. The data from the overexpression experiments were confirmed by SNAP-23 knockdown, which demonstrated a significant delay in phagosome maturation and a reduction in uptake activity. Finally, for analyzing whether phagosomal SNAP-23 entails a structural change in the protein, an intramolecular Förster resonance energy transfer (FRET) probe was constructed, in which the distance within a TagGFP2-TagRFP was altered upon close approximation of the N-termini of its two SNARE motifs. FRET efficiency on phagosomes was markedly enhanced only when VAMP7, a lysosomal SNARE, was coexpressed. Taken together, our results strongly suggest the involvement of SNAP-23 in both phagosome formation and maturation in macrophages, presumably by mediating SNARE-based membrane traffic.

Histone H1 induces bending and looping of single DNA molecules at nanomolar concentrations. H1 increases the rate of assembly for single chromatin fibers under force in Xenopus egg extracts and protects decondensing sperm nuclei from stretching and fragmenting in egg cytoplasm.

Histone H1 binds to linker DNA between nucleosomes, but the dynamics and biological ramifications of this interaction remain poorly understood. We performed single-molecule experiments using magnetic tweezers to determine the effects of H1 on naked DNA in buffer or during chromatin assembly in Xenopus egg extracts. In buffer, nanomolar concentrations of H1 induce bending and looping of naked DNA at stretching forces below 0.6 pN, effects that can be reversed with 2.7-pN force or in 200 mM monovalent salt concentrations. Consecutive tens-of-nanometer bending events suggest that H1 binds to naked DNA in buffer at high stoichiometries. In egg extracts, single DNA molecules assemble into nucleosomes and undergo rapid compaction. Histone H1 at endogenous physiological concentrations increases the DNA compaction rate during chromatin assembly under 2-pN force and decreases it during disassembly under 5-pN force. In egg cytoplasm, histone H1 protects sperm nuclei undergoing genome-wide decondensation and chromatin assembly from becoming abnormally stretched or fragmented due to astral microtubule pulling forces. These results reveal functional ramifications of H1 binding to DNA at the single-molecule level and suggest an important physiological role for H1 in compacting DNA under force and during chromatin assembly.

POS-1 and GLD-1 are required to repress the Notch homologue glp-1 translation in Caenorhabditis elegans embryos. Both proteins bind to overlapping elements in the glp-1 3' UTR. Two POS-1 sites are present within this region, but only one is required for repression in vivo. It is proposed that POS-1 restricts access of other RNA-binding proteins to this region.

RNA-binding proteins (RBPs) coordinate cell fate specification and differentiation in a variety of systems. RNA regulation is critical during oocyte development and early embryogenesis, in which RBPs control expression from maternal mRNAs encoding key cell fate determinants. The Caenorhabditis elegans Notch homologue glp-1 coordinates germline progenitor cell proliferation and anterior fate specification in embryos. A network of sequence-specific RBPs is required to pattern GLP-1 translation. Here, we map the cis-regulatory elements that guide glp-1 regulation by the CCCH-type tandem zinc finger protein POS-1 and the STAR-domain protein GLD-1. Our results demonstrate that both proteins recognize the glp-1 3′ untranslated region (UTR) through adjacent, overlapping binding sites and that POS-1 binding excludes GLD-1 binding. Both factors are required to repress glp-1 translation in the embryo, suggesting that they function in parallel regulatory pathways. It is intriguing that two equivalent POS-1–binding sites are present in the glp-1 3′ UTR, but only one, which overlaps with a translational derepression element, is functional in vivo. We propose that POS-1 regulates glp-1 mRNA translation by blocking access of other RBPs to a key regulatory sequence.