Functional redundancy and formin-independent localization of tropomyosin isoforms in Saccharomyces cerevisiae.

Tropomyosin is an actin binding protein which protects actin filaments from cofilin-mediated disassembly. Distinct tropomyosin isoforms have long been hypothesized to differentially sort to subcellular actin networks and impart distinct functionalities. Nevertheless, a mechanistic understanding of the interplay between Tpm isoforms and their functional contributions to actin dynamics has been lacking. In this study, we present acetylation-mimic engineered mNeonGreen-Tpm fusion proteins that exhibit complete functionality as a sole copy, surpassing limitations of existing probes and enabling real-time dynamic tracking of Tpm-actin filaments in vivo. Using these functional Tpm fusion proteins, we find that both Tpm1 and Tpm2 indiscriminately bind to actin filaments nucleated by either formin isoform- Bnr1 and Bni1 in vivo, in contrast to the long-held paradigm of Tpm-formin pairing. We also show that Tpm2 can protect and organize functional actin cables in absence of Tpm1. Overall, our work supports a concentration-dependent and formin-independent model of Tpm-actin binding and demonstrates for the first time, the functional redundancy of the paralog Tpm2 in actin cable maintenance in S. cerevisiae.

IntAct: A nondisruptive internal tagging strategy to study the organization and function of actin isoforms.

Mammals have 6 highly conserved actin isoforms with nonredundant biological functions. The molecular basis of isoform specificity, however, remains elusive due to a lack of tools. Here, we describe the development of IntAct, an internal tagging strategy to study actin isoforms in fixed and living cells. We identified a residue pair in ß-actin that permits tag integration and used knock-in cell lines to demonstrate that IntAct ß-actin expression and filament incorporation is indistinguishable from wild type. Furthermore, IntAct ß-actin remains associated with common actin-binding proteins (ABPs) and can be targeted in living cells. We demonstrate the usability of IntAct for actin isoform investigations by showing that actin isoform-specific distribution is maintained in human cells. Lastly, we observed a variant-dependent incorporation of tagged actin variants into yeast actin patches, cables, and cytokinetic rings demonstrating cross species applicability. Together, our data indicate that IntAct is a versatile tool to study actin isoform localization, dynamics, and molecular interactions.

A monomeric StayGold fluorescent protein.

StayGold is an exceptionally bright and stable fluorescent protein that is highly resistant to photobleaching. Despite favorable fluorescence properties, use of StayGold as a fluorescent tag is limited because it forms a natural dimer. Here we report the 1.6 Å structure of StayGold and generate a derivative, mStayGold, that retains the brightness and photostability of the original protein while being fully monomeric.

Evaluation of the Electrically-Evoked Stapedial Reflex Threshold in Pediatric Cochlear Implant Users with High-Frequency Probe Tones

Abstract Introduction Measurement of the electrically-evoked stapedial reflex threshold (ESRT) is an objective tool used to set the comfort levels in pediatric cochlear implant (PCI) users. The levels of ESRT have a strong correlation with comfort levels. However, the clinical utility of ESRT is limited because the ESRT response is not observed in all cochlear implant users. Objective To assess the effects of probe-tone frequency on ESRT and its relationship with the behavioral comfort levels in PCI users. Methods A total of 14 PCI users aged between 5 and 8 years participated in the study. The ESRT levels were measured using high-frequency probe tones (678 Hz and 1,000 Hz), and the default 226 Hz probe tone. The ESRT was measured with single-electrode stimulation across the three electrode locations (basal [E01]; middle [E11]; and apical [E22]). The ESRT levels measured with different probe tone frequencies were compared with the behavioral comfort levels. Results The mean ESRT levels using 1,000Hz and 678 Hz were lower than those measured using 226 Hz, but there was no main effect of probe-tone frequency (p > 0.05). A significantly high incidence of successful ESRT measurements occurred with higher-frequency probe tone (p < 0.039). Additionally, ESRT using higher probe tones significantly correlated with comfort levels. Conclusion The ESRT with higher probe tones was correlated with behavioral comfort levels and increased the success rate of the measurements. Higher-frequency probe tones may be useful whenever ESRT with 226 Hz is not measurable.

Evaluation of the Electrically-Evoked Stapedial Reflex Threshold in Pediatric Cochlear Implant Users with High-Frequency Probe Tones.

Introduction Measurement of the electrically-evoked stapedial reflex threshold (ESRT) is an objective tool used to set the comfort levels in pediatric cochlear implant (PCI) users. The levels of ESRT have a strong correlation with comfort levels. However, the clinical utility of ESRT is limited because the ESRT response is not observed in all cochlear implant users. Objective To assess the effects of probe-tone frequency on ESRT and its relationship with the behavioral comfort levels in PCI users. Methods A total of 14 PCI users aged between 5 and 8 years participated in the study. The ESRT levels were measured using high-frequency probe tones (678 Hz and 1,000 Hz), and the default 226 Hz probe tone. The ESRT was measured with single-electrode stimulation across the three electrode locations (basal [E01]; middle [E11]; and apical [E22]). The ESRT levels measured with different probe tone frequencies were compared with the behavioral comfort levels. Results The mean ESRT levels using 1,000Hz and 678 Hz were lower than those measured using 226 Hz, but there was no main effect of probe-tone frequency ( p > 0.05). A significantly high incidence of successful ESRT measurements occurred with higher-frequency probe tone ( p < 0.039). Additionally, ESRT using higher probe tones significantly correlated with comfort levels. Conclusion The ESRT with higher probe tones was correlated with behavioral comfort levels and increased the success rate of the measurements. Higher-frequency probe tones may be useful whenever ESRT with 226 Hz is not measurable.

ALIBY: ALFA Nanobody-Based Toolkit for Imaging and Biochemistry in Yeast.

Specialized epitope tags continue to be integral components of various biochemical and cell biological applications such as fluorescence microscopy, immunoblotting, immunoprecipitation, and protein purification. However, until recently, no single tag could offer this complete set of functionalities on its own. Here, we present a plasmid-based toolkit named ALIBY (ALFA toolkit for imaging and biochemistry in yeast) that provides a universal workflow to adopt the versatile ALFA tag/NbALFA system within the well-established model organism Saccharomyces cerevisiae. The kit comprises tagging plasmids for labeling a protein of interest with the ALFA tag and detection plasmids encoding fluorescent-protein-tagged NbALFA for live-cell imaging purposes. We demonstrate the suitability of ALIBY for visualizing the spatiotemporal localization of yeast proteins (i.e., the cytoskeleton, nucleus, centrosome, mitochondria, vacuole, endoplasmic reticulum, exocyst, and divisome) in live cells. Our approach has yielded an excellent signal-to-noise ratio without off-target effects or any effect on cell growth. In summary, our yeast-specific toolkit aims to simplify and further advance the live-cell imaging of differentially abundant yeast proteins while also being suitable for biochemical applications. IMPORTANCE In yeast research, conventional fluorescent protein tags and small epitope tags are widely used to study the spatiotemporal dynamics and activity of proteins. Although proven to be efficient, these tags lack the versatility for use across different cell biological and biochemical studies of a given protein of interest. Therefore, there is an urgent need for a unified platform for visualization and biochemical and functional analyses of proteins of interest in yeast. Here, we have engineered ALIBY, a plasmid-based toolkit that expands the benefits of the recently developed ALFA tag/NbALFA system to studies in the well-established model organism Saccharomyces cerevisiae. We demonstrate that ALIBY provides a simple and versatile strain construction workflow for long-duration live-cell imaging and biochemical applications in yeast.

mNG-tagged fusion proteins and nanobodies to visualize tropomyosins in yeast and mammalian cells.

Tropomyosins are structurally conserved α-helical coiled-coil proteins that bind along the length of filamentous actin (F-actin) in fungi and animals. Tropomyosins play essential roles in the stability of actin filaments and in regulating myosin II contractility. Despite the crucial role of tropomyosin in actin cytoskeletal regulation, in vivo investigations of tropomyosin are limited, mainly due to the suboptimal live-cell imaging tools currently available. Here, we report on an mNeonGreen (mNG)-tagged tropomyosin, with native promoter and linker length configuration, that clearly reports tropomyosin dynamics in Schizosaccharomyces pombe (Cdc8), Schizosaccharomyces japonicus (Cdc8) and Saccharomyces cerevisiae (Tpm1 and Tpm2). We also describe a fluorescent probe to visualize mammalian tropomyosin (TPM2 isoform). Finally, we generated a camelid nanobody against S. pombe Cdc8, which mimics the localization of mNG-Cdc8 in vivo. Using these tools, we report the presence of tropomyosin in previously unappreciated patch-like structures in fission and budding yeasts, show flow of tropomyosin (F-actin) cables to the cytokinetic actomyosin ring and identify rearrangements of the actin cytoskeleton during mating. These powerful tools and strategies will aid better analyses of tropomyosin and F-actin cables in vivo.

Asgard archaea shed light on the evolutionary origins of the eukaryotic ubiquitin-ESCRT machinery.

The ESCRT machinery, comprising of multiple proteins and subcomplexes, is crucial for membrane remodelling in eukaryotic cells, in processes that include ubiquitin-mediated multivesicular body formation, membrane repair, cytokinetic abscission, and virus exit from host cells. This ESCRT system appears to have simpler, ancient origins, since many archaeal species possess homologues of ESCRT-III and Vps4, the components that execute the final membrane scission reaction, where they have been shown to play roles in cytokinesis, extracellular vesicle formation and viral egress. Remarkably, metagenome assemblies of Asgard archaea, the closest known living relatives of eukaryotes, were recently shown to encode homologues of the entire cascade involved in ubiquitin-mediated membrane remodelling, including ubiquitin itself, components of the ESCRT-I and ESCRT-II subcomplexes, and ESCRT-III and Vps4. Here, we explore the phylogeny, structure, and biochemistry of Asgard homologues of the ESCRT machinery and the associated ubiquitylation system. We provide evidence for the ESCRT-I and ESCRT-II subcomplexes being involved in ubiquitin-directed recruitment of ESCRT-III, as it is in eukaryotes. Taken together, our analyses suggest a pre-eukaryotic origin for the ubiquitin-coupled ESCRT system and a likely path of ESCRT evolution via a series of gene duplication and diversification events.

Calponin-homology domain mediated bending of membrane-associated actin filaments.

Actin filaments are central to numerous biological processes in all domains of life. Driven by the interplay with molecular motors, actin binding and actin modulating proteins, the actin cytoskeleton exhibits a variety of geometries. This includes structures with a curved geometry such as axon-stabilizing actin rings, actin cages around mitochondria and the cytokinetic actomyosin ring, which are generally assumed to be formed by short linear filaments held together by actin cross-linkers. However, whether individual actin filaments in these structures could be curved and how they may assume a curved geometry remains unknown. Here, we show that 'curly', a region from the IQGAP family of proteins from three different organisms, comprising the actin-binding calponin-homology domain and a C-terminal unstructured domain, stabilizes individual actin filaments in a curved geometry when anchored to lipid membranes. Although F-actin is semi-flexible with a persistence length of ~10 µm, binding of mobile curly within lipid membranes generates actin filament arcs and full rings of high curvature with radii below 1 µm. Higher rates of fully formed actin rings are observed in the presence of the actin-binding coiled-coil protein tropomyosin and when actin is directly polymerized on lipid membranes decorated with curly. Strikingly, curly induced actin filament rings contract upon the addition of muscle myosin II filaments and expression of curly in mammalian cells leads to highly curved actin structures in the cytoskeleton. Taken together, our work identifies a new mechanism to generate highly curved actin filaments, which opens a range of possibilities to control actin filament geometries, that can be used, for example, in designing synthetic cytoskeletal structures.

Collective dynein transport of the nucleus by pulling on astral microtubules during Saccharomyces cerevisiae mitosis.

Positioning the nucleus at the bud neck during Saccharomyces cerevisiae mitosis involves pulling forces of cytoplasmic dynein localized in the daughter cell. Although genetic analysis has revealed a complex network positioning the nucleus, quantification of the forces acting on the nucleus and the number of dyneins driving the process has remained difficult. To better understand the collective forces involved in nuclear positioning, we compare a model of dyneins-driven microtubule (MT) pulling, MT pushing, and cytoplasmic drag to experiments. During S. cerevisiae mitosis, MTs interacting with the cortex nucleated by the daughter spindle pole body (SPB) (SPB-D) are longer than the mother SPB (SPB-M), increasing further during spindle elongation in anaphase. Interphasic SPB mobility is effectively diffusive, while the mitotic mobility is directed. By optimizing a computational model of the mobility of the nucleus due to diffusion and MTs pushing at the cell membrane to experiment, we estimate the viscosity governing the drag force on nuclei during positioning. A force balance model of mitotic SPB mobility compared to experimental mobility suggests that even one or two dynein dimers are sufficient to move the nucleus in the bud neck. Using stochastic computer simulations of a budding cell, we find that punctate dynein localization can generate sufficient force to reel in the nucleus to the bud neck. Compared to uniform motor localization, puncta involve fewer motors suggesting a functional role for motor clustering. Stochastic simulations also suggest that a higher number of force generators than predicted by force balance may be required to ensure the robustness of spindle positioning.

An organelle-tethering mechanism couples flagellation to cell division in bacteria.

In some free-living and pathogenic bacteria, problems in the synthesis and assembly of early flagellar components can cause cell-division defects. However, the mechanism that couples cell division with the flagellar biogenesis has remained elusive. Herein, we discover the regulator MadA that controls transcription of flagellar and cell-division genes in Caulobacter crescentus. We demonstrate that MadA, a small soluble protein, binds the type III export component FlhA to promote activation of FliX, which in turn is required to license the conserved σ54-dependent transcriptional activator FlbD. While in the absence of MadA, FliX and FlbD activation is crippled, bypass mutations in FlhA restore flagellar biogenesis and cell division. Furthermore, we demonstrate that MadA safeguards the divisome stoichiometry to license cell division. We propose that MadA has a sentinel-type function that senses an early flagellar biogenesis event and, through cell-division control, ensures that a flagellated offspring emerges.

Time-varying mobility and turnover of actomyosin ring components during cytokinesis in Schizosaccharomyces pombe.

Cytokinesis in many eukaryotes is dependent on a contractile actomyosin ring (AMR), composed of F-actin, myosin II, and other actin and myosin II regulators. Through fluorescence recovery after photobleaching experiments, many components of the AMR have been shown to be mobile and to undergo constant exchange with the cytosolic pools. However, how the mobility of its components changes at distinct stages of mitosis and cytokinesis has not been addressed. Here, we describe the mobility of eight Schizosaccharomyces pombe AMR proteins at different stages of mitosis and cytokinesis using an approach we have developed. We identified three classes of proteins, which showed 1) high (Ain1, Myo2, Myo51), 2) low (Rng2, Mid1, Myp2, Cdc12), and 3) cell cycle-dependent (Cdc15) mobile fractions. We observed that the F-BAR protein Cdc15 undergoes a 20-30% reduction in its mobile fraction after spindle breakdown and initiation of AMR contraction. Moreover, our data indicate that this change in Cdc15 mobility is dependent on the septation initiation network (SIN). Our work offers a novel strategy for estimating cell cycle-dependent mobile protein fractions in cellular structures and provides a valuable dataset, that is of interest to researchers working on cytokinesis.

Phosphoregulation of tropomyosin-actin interaction revealed using a genetic code expansion strategy.

Tropomyosins are coiled-coil proteins that regulate the stability and / or function of actin cytoskeleton in muscle and non-muscle cells through direct binding of actin filaments. Recently, using the fission yeast, we discovered a new mechanism by which phosphorylation of serine 125 of tropomyosin (Cdc8), reduced its affinity for actin filaments thereby providing access for the actin severing protein Adf1/Cofilin to actin filaments causing instability of actin filaments. Here we use a genetic code expansion strategy to directly examine this conclusion. We produced in Escherichia coli Cdc8-tropomyosin bearing a phosphate group on Serine-125 (Cdc8 PS125), using an orthogonal tRNA-tRNA synthetase pair that directly incorporates phosphoserine into proteins in response to a UAG codon in the corresponding mRNA. We show using total internal reflection (TIRF) microscopy that, whereas E.coli produced Cdc8 PS125 does not bind actin filaments, Cdc8 PS125 incubated with lambda phosphatase binds actin filaments. This work directly demonstrates that a phosphate moiety present on serine 125 leads to decreased affinity of Cdc8-tropomyosin for actin filaments. We also extend the work to demonstrate the usefulness of the genetic code expansion approach in imaging actin cytoskeletal components.

Genetic suppression of defective profilin by attenuated Myosin II reveals a potential role for Myosin II in actin dynamics in vivo in fission yeast.

The actin cytoskeleton plays a variety of roles in eukaryotic cell physiology, ranging from cell polarity and migration to cytokinesis. Key to the function of the actin cytoskeleton is the mechanisms that control its assembly, stability, and turnover. Through genetic analyses in Schizosaccharomyces pombe, we found that myo2-S1 (myo2-G515D), a Myosin II mutant allele, was capable of rescuing lethality caused by partial defects in actin nucleation/stability caused, for example, through compromised function of the actin-binding protein Cdc3-profilin. The mutation in myo2-S1 affects the activation loop of Myosin II, which is involved in physical interaction with subdomain 1 of actin and in stimulating the ATPase activity of Myosin. Consistently, actomyosin rings in myo2-S1 cell ghosts were unstable and severely compromised in contraction on ATP addition. These studies strongly suggest a role for Myo2 in actin cytoskeletal disassembly and turnover in vivo, and that compromise of this activity leads to genetic suppression of mutants defective in actin filament assembly/stability at the division site.

Measuring Binaural Temporal-Fine-Structure Sensitivity in Hearing-Impaired Listeners, Using the TFS-AF Test.

BACKGROUND: Various methods have been used to measure temporal-fine-structure (TFS) sensitivity in hearing-impaired (HI) listeners. A new method called TFS-adaptive frequency (TFS-AF) test, tracks the highest frequency up to which a person can detect a given interaural phase difference (IPD) in bursts of pure tones. So far, the test was only administered to listeners with normal hearing (NH) or impaired low-frequency hearing. It is currently not known if this test can also be used for listeners with different configurations of hearing losses. PURPOSE: To investigate whether the TFS-AF test can also be used on listeners with a larger diversity of hearing losses and what would be the best fixed IPD value to conduct the test. RESEARCH DESIGN: Using a cross-sectional study design, we compared the thresholds of TFS-AF test between the NH and HI listeners at three different IPDs (90°, 60°, and 30°). STUDY SAMPLE: Thirty NH (mean age = 37.9; range 19-53 years) and thirty HI (mean age = 38.6; range 19-53 years) with different configurations of hearing losses were tested. RESULTS: The listeners were able to complete the TFS-AF test at larger values of IPD. Average thresholds were lower (i.e., worse) in the HI listeners than in the NH listeners. Threshold did not correlate with the listeners' age in each group. CONCLUSION: This test can be used clinically as it provides a graded measure of TFS ability for young to young-old adult listeners with a variety of hearing losses.

Phosphoregulation of tropomyosin is crucial for actin cable turnover and division site placement.

Tropomyosin is a coiled-coil actin binding protein key to the stability of actin filaments. In muscle cells, tropomyosin is subject to calcium regulation, but its regulation in nonmuscle cells is not understood. Here, we provide evidence that the fission yeast tropomyosin, Cdc8, is regulated by phosphorylation of a serine residue. Failure of phosphorylation leads to an increased number and stability of actin cables and causes misplacement of the division site in certain genetic backgrounds. Phosphorylation of Cdc8 weakens its interaction with actin filaments. Furthermore, we show through in vitro reconstitution that phosphorylation-mediated release of Cdc8 from actin filaments facilitates access of the actin-severing protein Adf1 and subsequent filament disassembly. These studies establish that phosphorylation may be a key mode of regulation of nonmuscle tropomyosins, which in fission yeast controls actin filament stability and division site placement.

Expanding the Zebrafish Genetic Code through Site-Specific Introduction of Azido-lysine, Bicyclononyne-lysine, and Diazirine-lysine.

Site-specific incorporation of un-natural amino acids (UNAA) is a powerful approach to engineer and understand protein function. Site-specific incorporation of UNAAs is achieved through repurposing the amber codon (UAG) as a sense codon for the UNAA, using a tRNACUA that base pairs with an UAG codon in the mRNA and an orthogonal amino-acyl tRNA synthetase (aaRS) that charges the tRNACUA with the UNAA. Here, we report an expansion of the zebrafish genetic code to incorporate the UNAAs, azido-lysine (AzK), bicyclononyne-lysine (BCNK), and diazirine-lysine (AbK) into green fluorescent protein (GFP) and glutathione-s-transferase (GST). We also present proteomic evidence for UNAA incorporation into GFP. Our work sets the stage for the use of AzK, BCNK, and AbK introduction into proteins as a means to investigate and engineer their function in zebrafish.

Rapid production of pure recombinant actin isoforms in Pichia pastoris.

Actins are major eukaryotic cytoskeletal proteins, and they are involved in many important cell functions, including cell division, cell polarity, wound healing and muscle contraction. Despite obvious drawbacks, muscle actin, which is easily purified, is used extensively for biochemical studies of the non-muscle actin cytoskeleton. Here, we report a rapid and cost-effective method to purify heterologous actins expressed in the yeast Pichia pastoris Actin is expressed as a fusion with the actin-binding protein thymosin ß4 and purified by means of an affinity tag introduced in the fusion. Following cleavage of thymosin ß4 and the affinity tag, highly purified functional full-length actin is liberated. We purify actins from Saccharomycescerevisiae and Schizosaccharomycespombe, and the ß- and γ-isoforms of human actin. We also report a modification of the method that facilitates expression and purification of arginylated actin, a form of actin thought to regulate dendritic actin networks in mammalian cells. The methods we describe can be performed in all laboratories equipped for molecular biology, and should greatly facilitate biochemical and cell biological studies of the actin cytoskeleton.

Steric hindrance in the upper 50†kDa domain of the motor Myo2p leads to cytokinesis defects in fission yeast.

Cytokinesis in many eukaryotes requires a contractile actomyosin ring that is placed at the division site. In fission yeast, which is an attractive organism for the study of cytokinesis, actomyosin ring assembly and contraction requires the myosin II heavy chain Myo2p. Although myo2-E1, a temperature-sensitive mutant defective in the upper 50 kDa domain of Myo2p, has been studied extensively, the molecular basis of the cytokinesis defect is not understood. Here, we isolate myo2-E1-Sup2, an intragenic suppressor that contains the original mutation in myo2-E1 (G345R) and a second mutation in the upper 50 kDa domain (Y297C). Unlike myo2-E1-Sup1, a previously characterized myo2-E1 suppressor, myo2-E1-Sup2 reverses actomyosin ring contraction defects in vitro and in vivo Structural analysis of available myosin motor domain conformations suggests that a steric clash in myo2-E1, which is caused by the replacement of a glycine with a bulky arginine, is relieved in myo2-E1-Sup2 by mutation of a tyrosine to a smaller cysteine. Our work provides insight into the function of the upper 50 kDa domain of Myo2p, informs a molecular basis for the cytokinesis defect in myo2-E1, and may be relevant to the understanding of certain cardiomyopathies.

Actin turnover maintains actin filament homeostasis during cytokinetic ring contraction.

Cytokinesis in many eukaryotes involves a tension-generating actomyosin-based contractile ring. Many components of actomyosin rings turn over during contraction, although the significance of this turnover has remained enigmatic. Here, using Schizosaccharomyces japonicus, we investigate the role of turnover of actin and myosin II in its contraction. Actomyosin ring components self-organize into ∼1-µm-spaced clusters instead of undergoing full-ring contraction in the absence of continuous actin polymerization. This effect is reversed when actin filaments are stabilized. We tested the idea that the function of turnover is to ensure actin filament homeostasis in a synthetic system, in which we abolished turnover by fixing rings in cell ghosts with formaldehyde. We found that these rings contracted fully upon exogenous addition of a vertebrate myosin. We conclude that actin turnover is required to maintain actin filament homeostasis during ring contraction and that the requirement for turnover can be bypassed if homeostasis is achieved artificially.