Two-point optical manipulation reveals mechanosensitive remodeling of cell-cell contacts in vivo.

Biological tissues acquire reproducible shapes during development through dynamic cell behaviors. Most of these behaviors involve the remodeling of cell-cell contacts. During epithelial morphogenesis, contractile actomyosin networks remodel cell-cell contacts by shrinking and extending junctions between lateral cell surfaces. However, actomyosin networks not only generate mechanical stresses but also respond to them, confounding our understanding of how mechanical stresses remodel cell-cell contacts. Here, we develop a two-point optical manipulation method to impose different stress patterns on cell-cell contacts in the early epithelium of the Drosophila embryo. The technique allows us to produce junction extension and shrinkage through different push and pull manipulations at the edges of junctions. We use these observations to expand classical vertex-based models of tissue mechanics, incorporating negative and positive mechanosensitive feedback depending on the type of remodeling. In particular, we show that Myosin-II activity responds to junction strain rate and facilitates full junction shrinkage. Altogether our work provides insight into how stress produces efficient deformation of cell-cell contacts in vivo and identifies unanticipated mechanosensitive features of their remodeling.

MYO1F regulates antifungal immunity by regulating acetylation of microtubules.

Opportunistic fungal infections have become one of the leading causes of death among immunocompromised patients, resulting in an estimated 1.5 million deaths each year worldwide. The molecular mechanisms that promote host defense against fungal infections remain elusive. Here, we find that Myosin IF (MYO1F), an unconventional myosin, promotes the expression of genes that are critical for antifungal innate immune signaling and proinflammatory responses. Mechanistically, MYO1F is required for dectin-induced α-tubulin acetylation, acting as an adaptor that recruits both the adaptor AP2A1 and α-tubulin N-acetyltransferase 1 to α-tubulin; in turn, these events control the membrane-to-cytoplasm trafficking of spleen tyrosine kinase and caspase recruitment domain-containing protein 9 Myo1f-deficient mice are more susceptible than their wild-type counterparts to the lethal sequelae of systemic infection with Candida albicans Notably, administration of Sirt2 deacetylase inhibitors, namely AGK2, AK-1, or AK-7, significantly increases the dectin-induced expression of proinflammatory genes in mouse bone marrow-derived macrophages and microglia, thereby protecting mice from both systemic and central nervous system C. albicans infections. AGK2 also promotes proinflammatory gene expression in human peripheral blood mononuclear cells after Dectin stimulation. Taken together, our findings describe a key role for MYO1F in promoting antifungal immunity by regulating the acetylation of α-tubulin and microtubules, and our findings suggest that Sirt2 deacetylase inhibitors may be developed as potential drugs for the treatment of fungal infections.

SH3P2 suppresses osteoclast differentiation through restricting membrane localization of myosin 1E.

Osteoclasts are multinucleated cells responsible for bone resorption. Src homology 3 (SH3) domain-containing protein-2 (SH3P2)/osteoclast-stimulating factor-1 regulates osteoclast differentiation, but its exact role remains elusive. Here, we show that SH3P2 suppresses osteoclast differentiation. SH3P2 knockout (KO) mice displayed decreased femoral trabecular bone mass and enhanced localization of osteoclasts on the tibial trabecular bone surface, suggesting that SH3P2 suppresses bone resorption by osteoclasts. Osteoclast differentiation based on cellular multinuclearity induced by macrophage colony-stimulating factor and receptor activator of nuclear factor-κB ligand (RANKL) was enhanced in bone marrow-derived macrophages lacking SH3P2. RANKL induced SH3P2 dephosphorylation, which increased the association of actin-dependent motor protein myosin 1E (Myo1E) with SH3P2 and thereby prevented Myo1E localization to the plasma membrane. Consistent with this, Myo1E in the membrane fraction increased in SH3P2-KO cells. Together with the attenuated osteoclast differentiation in Myo1E knocked down cells, SH3P2 may suppress osteoclast differentiation by preventing their cell-to-cell fusion depending on Myo1E membrane localization.

The fungal myosin I is essential for Fusarium toxisome formation.

Myosin-I molecular motors are proposed to function as linkers between membranes and the actin cytoskeleton in several cellular processes, but their role in the biosynthesis of fungal secondary metabolites remain elusive. Here, we found that the myosin I of Fusarium graminearum (FgMyo1), the causal agent of Fusarium head blight, plays critical roles in mycotoxin biosynthesis. Inhibition of myosin I by the small molecule phenamacril leads to marked reduction in deoxynivalenol (DON) biosynthesis. FgMyo1 also governs translation of the DON biosynthetic enzyme Tri1 by interacting with the ribosome-associated protein FgAsc1. Disruption of the ATPase activity of FgMyo1 either by the mutation E420K, down-regulation of FgMyo1 expression or deletion of FgAsc1 results in reduced Tri1 translation. The DON biosynthetic enzymes Tri1 and Tri4 are mainly localized to subcellular structures known as toxisomes in response to mycotoxin induction and the FgMyo1-interacting protein, actin, participates in toxisome formation. The actin polymerization disruptor latrunculin A inhibits toxisome assembly. Consistent with this observation, deletion of the actin-associated proteins FgPrk1 and FgEnd3 also results in reduced toxisome formation. Unexpectedly, the FgMyo1-actin cytoskeleton is not involved in biosynthesis of another secondary metabolite tested. Taken together, this study uncovers a novel function of myosin I in regulating mycotoxin biosynthesis in filamentous fungi.

Fission yeast myosin I facilitates PI(4,5)P2-mediated anchoring of cytoplasmic dynein to the cortex.

Several key processes in the cell, such as vesicle transport and spindle positioning, are mediated by the motor protein cytoplasmic dynein, which produces force on the microtubule. For the functions that require movement of the centrosome and the associated nuclear material, dynein needs to have a stable attachment at the cell cortex. In fission yeast, Mcp5 is the anchor protein of dynein and is required for the oscillations of the horsetail nucleus during meiotic prophase. Although the role of Mcp5 in anchoring dynein to the cortex has been identified, it is unknown how Mcp5 associates with the membrane as well as the importance of the underlying attachment to the nuclear oscillations. Here, we set out to quantify Mcp5 organization and identify the binding partner of Mcp5 at the membrane. We used confocal and total internal reflection fluorescence microscopy to count the number of Mcp5 foci and the number of Mcp5 molecules in an individual focus. Further, we quantified the localization pattern of Mcp5 in fission yeast zygotes and show by perturbation of phosphatidylinositol 4-phosphate 5-kinase that Mcp5 binds to phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Remarkably, we discovered that the myosin I protein in fission yeast, Myo1, which is required for organization of sterol-rich domains in the cell membrane, facilitates the localization of Mcp5 and that of cytoplasmic dynein on the membrane. Finally, we demonstrate that Myo1-facilitated association of Mcp5 and dynein to the membrane determines the dynamics of nuclear oscillations and, in essence, dynein activity.

Genetic polymorphisms underlying the skeletal Class III phenotype.

INTRODUCTION: Our goal was to verify the association between candidate polymorphisms and skeletal Class III malocclusion in a well-characterized homogeneous sample set. METHODS: Thirty-five single-nucleotide polymorphisms were studied from 10 candidate loci in 54 Class III subjects and 120 controls. Skeletal Class III characteristics included ANB angle less than 0°, SNB angle greater than 83° (mandibular prognathism), SNA angle less than 79° (maxillary deficiency), Class III molar relationship, and negative overjet. Inclusion criteria for the controls were ANB angle between 0° and 4°, Class I molar relationship, and normal overjet. Chi-square and Fisher exact tests and principal component (PC) analysis were used to determine overrepresentation of marker alleles with alpha of 0.05. Odds ratios and 95% confidence intervals were calculated. RESULTS: MYO1H (rs10850110 AG) (P = 0.001) with PC2 and between FGF10 (rs593307 A

Brush border myosin Ia has tumor suppressor activity in the intestine.

The loss of the epithelial architecture and cell polarity/differentiation is known to be important during the tumorigenic process. Here we demonstrate that the brush border protein Myosin Ia (MYO1A) is important for polarization and differentiation of colon cancer cells and is frequently inactivated in colorectal tumors by genetic and epigenetic mechanisms. MYO1A frame-shift mutations were observed in 32% (37 of 116) of the colorectal tumors with microsatellite instability analyzed, and evidence of promoter methylation was observed in a significant proportion of colon cancer cell lines and primary colorectal tumors. The loss of polarization/differentiation resulting from MYO1A inactivation is associated with higher tumor growth in soft agar and in a xenograft model. In addition, the progression of genetically and carcinogen-initiated intestinal tumors was significantly accelerated in Myo1a knockout mice compared with Myo1a wild-type animals. Moreover, MYO1A tumor expression was found to be an independent prognostic factor for colorectal cancer patients. Patients with low MYO1A tumor protein levels had significantly shorter disease-free and overall survival compared with patients with high tumoral MYO1A (logrank test P = 0.004 and P = 0.009, respectively). The median time-to-disease recurrence in patients with low MYO1A was 1 y, compared with >9 y in the group of patients with high MYO1A. These results identify MYO1A as a unique tumor-suppressor gene in colorectal cancer and demonstrate that the loss of structural brush border proteins involved in cell polarity are important for tumor development.

Myosin IC generates power over a range of loads via a new tension-sensing mechanism.

Myosin IC (myo1c), a widely expressed motor protein that links the actin cytoskeleton to cell membranes, has been associated with numerous cellular processes, including insulin-stimulated transport of GLUT4, mechanosensation in sensory hair cells, endocytosis, transcription of DNA in the nucleus, exocytosis, and membrane trafficking. The molecular role of myo1c in these processes has not been defined, so to better understand myo1c function, we utilized ensemble kinetic and single-molecule techniques to probe myo1c's biochemical and mechanical properties. Utilizing a myo1c construct containing the motor and regulatory domains, we found the force dependence of the actin-attachment lifetime to have two distinct regimes: a force-independent regime at forces < 1 pN, and a highly force-dependent regime at higher loads. In this force-dependent regime, forces that resist the working stroke increase the actin-attachment lifetime. Unexpectedly, the primary force-sensitive transition is the isomerization that follows ATP binding, not ADP release as in other slow myosins. This force-sensing behavior is unique amongst characterized myosins and clearly demonstrates mechanochemical diversity within the myosin family. Based on these results, we propose that myo1c functions as a slow transporter rather than a tension-sensitive anchor.

Fibre type-specific satellite cell response to aerobic training in sedentary adults.

In the present study, we sought to determine the effect of a traditional, 12 week aerobic training protocol on skeletal muscle fibre type distribution and satellite cell content in sedentary subjects. Muscle biopsies were obtained from the vastus lateralis [n = 23 subjects (six male and 17 female); body mass index 30.7 ± 1.2 kg m(-2)] before and after 12 weeks of aerobic training performed on a cycle ergometer. Immunohistochemical analyses were used to quantify myosin heavy chain (MyHC) isoform expression, cross-sectional area and satellite cell and myonuclear content. Following training, a decrease in MyHC hybrid type IIa/IIx fibre frequency occurred, with a concomitant increase in pure MyHC type IIa fibres. Pretraining fibre type correlated with body mass index, and the change in fibre type following training was associated with improvements in maximal oxygen consumption. Twelve weeks of aerobic training also induced increases in mean cross-sectional area in both MyHC type I and type IIa fibres. Satellite cell content was also increased following training, specifically in MyHC type I fibres, with no change in the number of satellite cells associated with MyHC type II fibres. With the increased satellite cell content following training, an increase in myonuclear number per fibre also occurred in MyHC type I fibres. Hypertrophy of MyHC type II fibres occurred without detectable myonuclear addition, suggesting that the mechanisms underlying growth in fast and slow fibres differ. These data provide intriguing evidence for a fibre type-specific role of satellite cells in muscle adaptation following aerobic training.

Left-right asymmetry: class I myosins show the direction.

Myosins are actin-based molecular motors that are found in almost all eukaryotes. Phylogenetic analysis allows the discrimination of 37 different types of myosins, most with unknown functions. Recent work in Drosophila has revealed a crucial role for type ID unconventional myosin in left-right asymmetry. Mutations in Myosin ID completely reverse the left-right axis (situs inversus), a phenotype that is dependent on an intact actin cytoskeleton. How this myosin might orient the left-right axis has began to be elucidated by showing that it interacts directly with beta-catenin, suggesting that myosin ID interacts with the adherens junction to control the direction of organ looping. This is the first demonstration of a role of a myosin in body patterning.

Myosin 1G is an abundant class I myosin in lymphocytes whose localization at the plasma membrane depends on its ancient divergent pleckstrin homology (PH) domain (Myo1PH).

Class I myosins, which link F-actin to membrane, are largely undefined in lymphocytes. Mass spectrometric analysis of lymphocytes identified two short tail forms: (Myo1G and Myo1C) and one long tail (Myo1F). We investigated Myo1G, the most abundant in T-lymphocytes, and compared key findings with Myo1C and Myo1F. Myo1G localizes to the plasma membrane and associates in an ATP-releasable manner to the actin-containing insoluble pellet. The IQ+tail region of Myo1G (Myo1C and Myo1F) is sufficient for membrane localization, but membrane localization is augmented by the motor domain. The minimal region lacks IQ motifs but includes: 1) a PH-like domain; 2) a "Pre-PH" region; and 3) a "Post-PH" region. The Pre-PH predicted alpha helices may contribute electrostatically, because two conserved basic residues on one face are required for optimal membrane localization. Our sequence analysis characterizes the divergent PH domain family, Myo1PH, present also in long tail myosins, in eukaryotic proteins unrelated to myosins, and in a probable ancestral protein in prokaryotes. The Myo1G Myo1PH domain utilizes the classic lipid binding site for membrane association, because mutating either of two basic residues in the "signature motif" destroys membrane localization. Mutation of each basic residue of the Myo1G Myo1PH domain reveals another critical basic residue in the beta3 strand, which is shared only by Myo1D. Myo1G differs from Myo1C in its phosphatidylinositol 4,5-bisphosphate dependence for membrane association, because membrane localization of phosphoinositide 5-phosphatase releases Myo1C from the membrane but not Myo1G. Thus Myo1PH domains likely play universal roles in myosin I membrane association, but different isoforms have diverged in their binding specificity.

Organelle transport: dynamic actin tracks for myosin motors.

Transport of cargo by molecular motors on microtubule and actin filament tracks is a fundamental property of eukaryotic cells. A new study reports that actin dynamics are required in cells for myosin I and V motor proteins to transport their organelle cargos on actin tracks.

Myosin I and actin dynamics: the frogs weigh in.

In this issue of Developmental Cell, Sokac et al. (2006) describe an intriguing new role for an actin-based motor protein in restraining actin polymerization during endocytosis in Xenopus oocytes.

Endocytic internalization in budding yeast requires coordinated actin nucleation and myosin motor activity.

Actin polymerization essential for endocytic internalization in budding yeast is controlled by four nucleation promoting factors (NPFs) that each exhibits a unique dynamic behavior at endocytic sites. How each NPF functions and is regulated to restrict actin assembly to late stages of endocytic internalization is not known. Quantitative analysis of NPF biochemical activities, and genetic analysis of recruitment and regulatory mechanisms, defined a linear pathway in which protein composition changes at endocytic sites control actin assembly and function. We show that yeast WASP initiates actin assembly at endocytic sites and that this assembly and the recruitment of a yeast WIP-like protein by WASP recruit a type I myosin with both NPF and motor activities. Importantly, type I myosin motor and NPF activities are separable, and both contribute to endocytic coat inward movement, which likely represents membrane invagination. These results reveal a mechanism in which actin nucleation and myosin motor activity cooperate to promote endocytic internalization.

Myosin-1c couples assembling actin to membranes to drive compensatory endocytosis.

Compensatory endocytosis follows regulated exocytosis in cells ranging from eggs to neurons, but the means by which it is accomplished are unclear. In Xenopus eggs, compensatory endocytosis is driven by dynamic coats of assembling actin that surround and compress exocytosing cortical granules (CGs). We have identified Xenopus laevis myosin-1c (XlMyo1c) as a myosin that is upregulated by polyadenylation during meiotic maturation, the developmental interval that prepares eggs for fertilization and regulated CG exocytosis. Upon calcium-induced exocytosis, XlMyo1c is recruited to exocytosing CG membranes where actin coats then assemble. When XlMyo1c function is disrupted, actin coats assemble, but dynamic actin filaments are uncoupled from the exocytosing CG membranes such that coats do not compress, and compensatory endocytosis fails. Remarkably, there is also an increase in polymerized actin at membranes throughout the cell. We conclude that XlMyo1c couples polymerizing actin to membranes and so mediates force production during compensatory endocytosis.

In pancreatic ß-cells myosin 1b regulates glucose-stimulated insulin secretion by modulating an early step in insulin granule trafficking from the Golgi.

Pancreatic ß-cells secrete insulin, which controls blood glucose levels, and defects in insulin secretion are responsible for diabetes mellitus. The actin cytoskeleton and some myosins support insulin granule trafficking and release, although a role for the class I myosin Myo1b, an actin- and membrane-associated load-sensitive motor, in insulin biology is unknown. We found by immunohistochemistry that Myo1b is expressed in islet cells of the rat pancreas. In cultured rat insulinoma 832/13 cells, Myo1b localized near actin patches, the trans-Golgi network (TGN) marker TGN38, and insulin granules in the perinuclear region. Myo1b depletion by small interfering RNA in 832/13 cells reduced intracellular proinsulin and insulin content and glucose-stimulated insulin secretion (GSIS) and led to the accumulation of (pro)insulin secretory granules (SGs) at the TGN. Using an in situ fluorescent pulse-chase strategy to track nascent proinsulin, Myo1b depletion in insulinoma cells reduced the number of (pro)insulin-containing SGs budding from the TGN. The studies indicate for the first time that in pancreatic ß-cells Myo1b controls GSIS at least in part by mediating an early stage in insulin granule trafficking from the TGN.

Left-right asymmetry: actin-myosin through the looking glass.

Despite being bilaterally symmetric, most Metazoa exhibit clear, genetically determined left-right differences. In several animals, microtubule-based structures are thought to be the source of chiral information used to establish handedness. Now, two new studies in Drosophila identify a role for unconventional myosin motors in this process.

Opposing Kinesin and Myosin-I Motors Drive Membrane Deformation and Tubulation along Engineered Cytoskeletal Networks.

Microtubule and actin filament molecular motors such as kinesin-1 and myosin-Ic (Myo1c) transport and remodel membrane-bound vesicles; however, it is unclear how they coordinate to accomplish these tasks. We introduced kinesin-1- and Myo1c-bound giant unilamellar vesicles (GUVs) into a micropatterned in vitro cytoskeletal matrix modeled after the subcellular architecture where vesicular sorting and membrane remodeling are observed. This array was composed of sparse microtubules intersecting regions dense with actin filaments, and revealed that Myo1c-dependent tethering of GUVs enabled kinesin-1-driven membrane deformation and tubulation. Membrane remodeling at actin/microtubule intersections was modulated by lipid composition and the addition of the Bin-Amphiphysin-Rvs-domain (BAR-domain) proteins endophilin or FCH-domain-only (FCHo). Myo1c not only tethered microtubule-transported cargo, but also transported, deformed, and tubulated GUVs along actin filaments in a lipid-composition- and BAR-protein-responsive manner. These results suggest a mechanism for actin-based involvement in vesicular transport and remodeling of intracellular membranes, and implicate lipid composition as a key factor in determining whether vesicles will undergo transport, deformation, or tubulation driven by opposing actin and microtubule motors and BAR-domain proteins.

Dissection of Arp2/3 complex actin nucleation mechanism and distinct roles for its nucleation-promoting factors in Saccharomyces cerevisiae.

Actin nucleation by the Arp2/3 complex is under tight control, remaining inactive until stimulation by nucleation-promoting factors (NPFs). Although multiple NPFs are expressed in most cell types, little is known about how they are coordinated and whether they perform similar or distinct functions. We examined genetic relationships among the four S. cerevisiae NPFs. Combining las17delta with pan1-101 or myo3delta myo5delta was lethal at all temperatures, whereas combining pan1-101 with myo3delta myo5delta showed no genetic interaction and abp1delta partially suppressed las17delta. These data suggest that NPFs have distinct and overlapping functions in vivo. We also tested genetic interactions between each NPF mutant and seven different temperature-sensitive arp2 alleles and purified mutant Arp2/3 complexes to compare their activities. Two arp2 alleles with mutations at the barbed end were severely impaired in nucleation, providing the first experimental evidence that Arp2 nucleates actin at its barbed end in vitro and in vivo. Another arp2 allele caused partially unregulated ("leaky") nucleation in the absence of NPFs. Combining this mutant with a partially unregulated allele in a different subunit of Arp2/3 complex was lethal, suggesting that cells cannot tolerate high levels of unregulated activity. Genetic interactions between arp2 alleles and NPF mutants point to Abp1 having an antagonistic role with respect to other NPFs, possibly serving to attenuate their stronger activities. In support of this model, Abp1 binds strongly to Arp2/3 complex, yet has notably weak nucleation-promoting activity and inhibits Las17 activity on Arp2/3 complex in a dose-responsive manner.