Cytoskeletal diversification across 1 billion years: What red algae can teach us about the cytoskeleton, and vice versa.

The cytoskeleton has a central role in eukaryotic biology, enabling cells to organize internally, polarize, and translocate. Studying cytoskeletal machinery across the tree of life can identify common elements, illuminate fundamental mechanisms, and provide insight into processes specific to less-characterized organisms. Red algae represent an ancient lineage that is diverse, ecologically significant, and biomedically relevant. Recent genomic analysis shows that red algae have a surprising paucity of cytoskeletal elements, particularly molecular motors. Here, we review the genomic and cell biological evidence and propose testable models of how red algal cells might perform processes including cell motility, cytokinesis, intracellular transport, and secretion, given their reduced cytoskeletons. In addition to enhancing understanding of red algae and lineages that evolved from red algal endosymbioses (e.g., apicomplexan parasites), these ideas may also provide insight into cytoskeletal processes in animal cells.

Variable penetrance of Nab3 granule accumulation quantified by a new tool for high-throughput single-cell granule analysis.

Reorganization of cellular proteins into subcellular compartments, such as the concentration of RNA-binding proteins into cytoplasmic stress granules and P-bodies, is a well-recognized, widely studied physiological process currently under intense investigation. One example of this is the induction of the yeast Nab3 transcription termination factor to rearrange from its pan-nucleoplasmic distribution to a granule at the nuclear periphery in response to nutrient limitation. Recent work in many cell types has shown that protein condensation in the nucleus is functionally important for transcription initiation, RNA processing, and termination. However, little is known about how subnuclear compartments form. Here, we have quantitatively analyzed this dynamic process in living yeast using a high-throughput computational tool and fluorescence microscopy. This analysis revealed that Nab3 granule accumulation varies in penetrance across yeast strains. A concentrated single granule is formed from at least a quarter of the nuclear Nab3 drawn from the rest of the nucleus. Levels of granule accumulation were inversely correlated with a growth defect in the absence of glucose. Importantly, the basis for some of the variation in penetrance was attributable to a defect in mitochondrial function. This publicly available computational tool provides a rigorous, reproducible, and unbiased examination of Nab3 granule accumulation that should be widely applicable to a variety of fluorescent images. Thousands of live cells can be readily examined enabling rigorous statistical verification of significance. With it, we describe a new feature of inducible subnuclear compartment formation for RNA-binding transcription factors and an important determinant of granule biogenesis.

Redundant Trojan horse and endothelial-circulatory mechanisms for host-mediated spread of Candida albicans yeast.

The host innate immune system has developed elegant processes for the detection and clearance of invasive fungal pathogens. These strategies may also aid in the spread of pathogens in vivo, although technical limitations have previously hindered our ability to view the host innate immune and endothelial cells to probe their roles in spreading disease. Here, we have leveraged zebrafish larvae as a model to view the interactions of these host processes with the fungal pathogen Candida albicans in vivo. We examined three potential host-mediated mechanisms of fungal spread: movement inside phagocytes in a "Trojan Horse" mechanism, inflammation-assisted spread, and endothelial barrier passage. Utilizing both chemical and genetic tools, we systematically tested the loss of neutrophils and macrophages and the loss of blood flow on yeast cell spread. Both neutrophils and macrophages respond to yeast-locked and wild type C. albicans in our model and time-lapse imaging revealed that macrophages can support yeast spread in a "Trojan Horse" mechanism. Surprisingly, loss of immune cells or inflammation does not alter dissemination dynamics. On the other hand, when blood flow is blocked, yeast can cross into blood vessels but they are limited in how far they travel. Blockade of both phagocytes and circulation reduces rates of dissemination and significantly limits the distance of fungal spread from the infection site. Together, this data suggests a redundant two-step process whereby (1) yeast cross the endothelium inside phagocytes or via direct uptake, and then (2) they utilize blood flow or phagocytes to travel to distant sites.

Cellular noise suppression by the regulator of G protein signaling Sst2.

G proteins and their associated receptors process information from a variety of environmental stimuli to induce appropriate cellular responses. Generally speaking, each cell in a population responds within defined limits, despite large variation in the expression of protein signaling components. Therefore, we postulated that noise suppression is encoded within the signaling system. Using the yeast mating pathway as a model, we evaluated the ability of a regulator of G protein signaling (RGS) protein to suppress noise. We found that the RGS protein Sst2 limits variability in transcription and morphogenesis in response to pheromone stimulation. While signal suppression is a result of both the GAP (GTPase accelerating) and receptor binding functions of Sst2, noise suppression requires only the GAP activity. Taken together, our findings reveal a hitherto overlooked role of RGS proteins as noise suppressors and demonstrate an ability to uncouple signal and noise in a prototypical stimulus-response pathway.

Systematic analysis of F-box proteins reveals a new branch of the yeast mating pathway.

The mating pathway in yeast Saccharomyces cerevisiae has long been used to reveal new mechanisms of signal transduction. The pathway comprises a pheromone receptor, a heterotrimeric G protein, and intracellular effectors of morphogenesis and transcription. Polarized cell growth, in the direction of a potential mating partner, is accomplished by the G-protein ßγ subunits and the small G-protein Cdc42. Transcription induction, needed for cell-cell fusion, is mediated by Gßγ and the mitogen-activated protein kinase (MAPK) scaffold protein Ste5. A potential third pathway is initiated by the G-protein α subunit Gpa1. Gpa1 signaling was shown previously to involve the F-box adaptor protein Dia2 and an endosomal effector protein, the phosphatidylinositol 3-kinase Vps34. Vps34 is also required for proper vacuolar sorting and autophagy. Here, using a panel of reporter assays, we demonstrate that mating pheromone stimulates vacuolar targeting of a cytoplasmic reporter protein and that this process depends on Vps34. Through a systematic analysis of F-box deletion mutants, we show that Dia2 is required to sustain pheromone-induced vacuolar targeting. We also found that other F-box proteins selectively regulate morphogenesis (Ydr306, renamed Pfu1) and transcription (Ucc1). These findings point to the existence of a new and distinct branch of the pheromone-signaling pathway, one that likely leads to vacuolar engulfment of cytoplasmic proteins and recycling of cellular contents in preparation for mating.

Triclosan disrupts immune cell function by depressing Ca2+ influx following acidification of the cytoplasm.

Triclosan (TCS) is an antimicrobial agent that was effectively banned by the FDA from hand soaps in 2016, hospital soaps in 2017, and hand sanitizers in 2019; however, TCS can still be found in a few products. At consumer-relevant, non-cytotoxic doses, TCS inhibits the functions of both mitochondria and mast cells, a ubiquitous cell type. Via the store-operated Ca2+ entry mechanism utilized by many immune cells, mast cells undergo antigen-stimulated Ca2+ influx into the cytosol, for proper function. Previous work showed that TCS inhibits Ca2+ dynamics in mast cells, and here we show that TCS also inhibits Ca2+ mobilization in human Jurkat T cells. However, the biochemical mechanism behind the Ca2+ dampening has yet to be elucidated. Three-dimensional super-resolution microscopy reveals that TCS induces mitochondrial swelling, in line with and extending the previous finding of TCS inhibition of mitochondrial membrane potential via its proton ionophoric activity. Inhibition of plasma membrane potential (PMP) by the canonical depolarizer gramicidin can inhibit mast cell function. However, use of the genetically encoded voltage indicators (GEVIs) ArcLight (pH-sensitive) and ASAP2 (pH-insensitive), indicates that TCS does not disrupt PMP. In conjunction with data from a plasma membrane-localized, pH-sensitive reporter, these results indicate that TCS, instead, induces cytosolic acidification in mast cells and T cells. Acidification of the cytosol likely inhibits Ca2+ influx by uncoupling the STIM1/ORAI1 interaction that is required for opening of plasma membrane Ca2+ channels. These results provide a mechanistic explanation of TCS disruption of Ca2+ influx and, thus, of immune cell function.

Fluorescence-based quantification of nucleocytoplasmic transport.

The sequestration of DNA within the membrane-bound nucleus is a defining characteristic of eukaryotic cells. Replication and transcription are therefore restricted to the nucleus, however, the regulation of these events relies on cytoplasmic processes including protein synthesis and signal transduction pathways. Because a variety of cellular activities depend on nuclear transport, researchers from diverse fields have found it useful to examine the nuclear localization of proteins of interest. Here we present some important technical considerations for studying nuclear and cytoplasmic localization, and provide guidance for quantifying protein levels using fluorescence microscopy and ImageJ software. We include discussion of the use of regions of interest and image segmentation for quantification of protein localization. Nucleocytoplasmic transport is fundamentally important for controlling protein levels and activity in the nucleus or cytoplasm, and quantitative analysis can provide insight into how biological output is achieved.

TOR signaling regulates GPCR levels on the plasma membrane and suppresses the Saccharomyces cerevisiae mating pathway.

Saccharomyces cerevisiae respond to mating pheromone through the GPCRs Ste2 and Ste3, which promote growth of a mating projection in response to ligand binding. This commitment to mating is nutritionally and energetically taxing, and so we hypothesized that the cell may suppress mating signaling during starvation. We set out to investigate negative regulators of the mating pathway in nutritionally depleted environments. Here, we report that nutrient deprivation led to loss of Ste2 from the plasma membrane. Recapitulating this effect with nitrogen starvation led us to hypothesize that it was due to TORC1 signaling. Rapamycin inhibition of TORC1 impacted membrane levels of all yeast GPCRs. Inhibition of TORC1 also dampened mating pathway output. Deletion analysis revealed that TORC1 repression leads to α-arrestin-directed CME through TORC2-Ypk1 signaling. We then set out to determine whether major downstream effectors of the TOR complexes also downregulate pathway output during mating. We found that autophagy contributes to pathway downregulation through analysis of strains lacking ATG8 . We also show that Ypk1 significantly reduced pathway output. Thus, both autophagy machinery and TORC2-Ypk1 signaling serve as attenuators of pheromone signaling during mating. Altogether, we demonstrate that the stress-responsive TOR complexes coordinate GPCR endocytosis and reduce the magnitude of pheromone signaling, in ligand-independent and ligand-dependent contexts. One Sentence Summary: TOR signaling regulates the localization of all Saccharomyces cerevisiae GPCRs during starvation and suppress the mating pathway in the presence and absence of ligand.

The roles of yeast formins and their regulators Bud6 and Bil2 in the pheromone response.

In response to pheromone Saccharomyces cerevisiae extend a mating projection. This process depends on the formation of polarized actin cables which direct secretion to the mating tip and translocate the nucleus for karyogamy. Here, we demonstrate that proper mating projection formation requires the formin Bni1, as well as the actin nucleation promoting activities of Bud6, but not the formin Bnr1. Further, Bni1 is required for pheromone gradient tracking. Our work also reveals unexpected new functions for Bil2 in the pheromone response. Previously we identified Bil2 as a direct inhibitor of Bnr1 during vegetative cell growth. Here, we show that Bil2 has Bnr1-independent functions in spatially focusing Bni1-GFP at mating projection tips, and in vitro Bil2 and its binding partner Bud6 organize Bni1 into clusters that nucleate actin assembly. bil2∆ cells also display entangled Bni1-generated actin cable arrays and defects in secretory vesicle transport and nuclear positioning. At low pheromone concentrations, bil2∆ cells are delayed in establishing a polarity axis, and at high concentrations they prematurely form a second and a third mating projection. Together, these results suggest that Bil2 promotes the proper formation and timing of mating projections by organizing Bni1 and maintaining a persistent axis of polarized growth.

Fast-twitch myofibrils grow in proportion to Mylpf dosage in the zebrafish embryo.

Muscle cells become stronger by expanding myofibrils, the chains of sarcomeres that produce contraction. Here we investigate how Mylpf (Myosin Light Chain Phosphorylatable Fast) abundance impacts myofibril assembly in fast-twitch muscle. The two zebrafish Mylpf genes (mylpfa and mylpfb) are exclusively expressed in fast-twitch muscle. We show that these cells initially produce six times more mylpfa mRNA and protein than mylpfb. The combined Mylpf protein dosage is necessary for and proportionate to fast-twitch myofibril growth in the embryo. Fast-twitch myofibrils are severely reduced in the mylpfa -/- mutant, leading to loss of high-speed movement; however, by persistent slow movement this mutant swims as far through time as its wild-type sibling. Although the mylpfb -/- mutant has normal myofibrils, myofibril formation fails entirely in the mylpfa -/- ;mylpfb -/- double mutant, indicating that the two genes are collectively essential to myofibril formation. Fast-twitch myofibril width is restored in the mylpfa -/- mutant by transgenic expression of mylpfa-GFP, mylpfb-GFP, and by human MYLPF-GFP to a degree corresponding linearly with GFP brightness. This correlate is inverted by expression of MYLPF alleles that cause Distal Arthrogryposis, which reduce myofibril size in proportion to protein abundance. These effects indicate that Mylpf dosage controls myofibril growth, impacting embryonic development and lifelong health.

The G-alpha Gpa1 directs septin localization in the mating projection of Saccharomyces cerevisiae through its Ubiquitination Domain and Endocytic Machinery.

The yeast mating response uses a G-protein coupled receptor (GPCR), Ste2, to detect mating pheromone and initiate mating projection morphogenesis. The septin cytoskeleton plays a key role in the formation of the mating projection, forming structures at the base of the projection. Desensitization of the Gα, Gpa1, by the Regulator of G-protein Signaling (RGS), Sst2, is required for proper septin organization and morphogenesis. In cells where the Gα is hyperactive, septins are mislocalized to the site of polarity, and the cells are unable to track a pheromone gradient. We set out to identify the proteins that mediate Gα control of septins during the Saccharomyces cerevisiae mating response by making mutations to rescue septin localization in cells expressing the hyperactive Gα mutant gpa1G302S. We found that single deletions of the septin chaperone Gic1, the Cdc42 GAP Bem3, and the epsins Ent1 and Ent2 rescued the polar cap accumulation of septins in the hyperactive Gα. We created an agent-based model of vesicle trafficking that predicts how changes in endocytic cargo licensing alters localization of endocytosis that mirrors the septin localization we see experimentally. We hypothesized that hyperactive Gα may increase the rate of endocytosis of a pheromone responsive cargo, thereby altering where septins are localized. Both the GPCR and the Gα are known to be internalized by clathrin-mediated endocytosis during the pheromone response. Deletion of the GPCR C-terminus to block internalization partially rescued septin organization. However, deletion of the Gpa1 ubiquitination domain required for its endocytosis completely abrogated septin accumulation at the polarity site. Our data support a model where the location of endocytosis serves as a spatial mark for septin structure assembly and that desensitization of the Gα delays its endocytosis sufficiently that septins are placed peripheral to the site of Cdc42 polarity.

Pheromone- and RSP5-dependent ubiquitination of the G protein beta subunit Ste4 in yeast.

Ste4 is the ß subunit of a heterotrimeric G protein that mediates mating responses in Saccharomyces cerevisiae. Here we show that Ste4 undergoes ubiquitination in response to pheromone stimulation. Ubiquitination of Ste4 is dependent on the E3 ligase Rsp5. Disrupting the activity of Rsp5 abolishes ubiquitination of Ste4 in vivo, and recombinant Rsp5 is capable of ubiquitinating Ste4 in vitro. We find also that Lys-340 is a major ubiquitination site on Ste4, as pheromone-induced ubiquitination of the protein is prevented when this residue is mutated to an arginine. Functionally, ubiquitination does not appear to regulate the stability of Ste4, as blocking ubiquitination has no apparent effect on either the abundance or the half-life of the protein. However, when presented with a concentration gradient of pheromone, Ste4(K340R) mutant cells polarize significantly faster than wild-type cells, indicating that ubiquitination limits pheromone-directed polarized growth. Together, these findings reveal a novel stimulus-dependent posttranslational modification of a Gß subunit, establish Ste4 as a new substrate of the E3 ligase Rsp5, and demonstrate a role for G protein ubiquitination in cell polarization.

Phosphorylation of RGS regulates MAP kinase localization and promotes completion of cytokinesis.

Yeast use the G-protein-coupled receptor signaling pathway to detect and track the mating pheromone. The G-protein-coupled receptor pathway is inhibited by the regulator of G-protein signaling (RGS) Sst2 which induces Gα GTPase activity and inactivation of downstream signaling. G-protein signaling activates the MAPK Fus3, which phosphorylates the RGS; however, the role of this modification is unknown. We found that pheromone-induced RGS phosphorylation peaks early; the phospho-state of RGS controls its localization and influences MAPK spatial distribution. Surprisingly, phosphorylation of the RGS promotes completion of cytokinesis before pheromone-induced growth. Completion of cytokinesis in the presence of pheromone is promoted by the kelch-repeat protein, Kel1 and antagonized by the formin Bni1. We found that RGS complexes with Kel1 and prefers the unphosphorylatable RGS mutant. We also found overexpression of unphosphorylatable RGS exacerbates cytokinetic defects, whereas they are rescued by overexpression of Kel1. These data lead us to a model where Kel1 promotes completion of cytokinesis before pheromone-induced polarity but is inhibited by unphosphorylated RGS binding.

Beneficial impacts of neuromuscular electrical stimulation on muscle structure and function in the zebrafish model of Duchenne muscular dystrophy.

Neuromuscular electrical stimulation (NMES) allows activation of muscle fibers in the absence of voluntary force generation. NMES could have the potential to promote muscle homeostasis in the context of muscle disease, but the impacts of NMES on diseased muscle are not well understood. We used the zebrafish Duchenne muscular dystrophy (dmd) mutant and a longitudinal design to elucidate the consequences of NMES on muscle health. We designed four neuromuscular stimulation paradigms loosely based on weightlifting regimens. Each paradigm differentially affected neuromuscular structure, function, and survival. Only endurance neuromuscular stimulation (eNMES) improved all outcome measures. We found that eNMES improves muscle and neuromuscular junction morphology, swimming, and survival. Heme oxygenase and integrin alpha7 are required for eNMES-mediated improvement. Our data indicate that neuromuscular stimulation can be beneficial, suggesting that the right type of activity may benefit patients with muscle disease.

Temporal dynamics of viral load and false negative rate influence the levels of testing necessary to combat COVID-19 spread.

Colleges and other organizations are considering testing plans to return to operation as the COVID-19 pandemic continues. Pre-symptomatic spread and high false negative rates for testing may make it difficult to stop viral spread. Here, we develop a stochastic agent-based model of COVID-19 in a university sized population, considering the dynamics of both viral load and false negative rate of tests on the ability of testing to combat viral spread. Reported dynamics of SARS-CoV-2 can lead to an apparent false negative rate from ~ 17 to ~ 48%. Nonuniform distributions of viral load and false negative rate lead to higher requirements for frequency and fraction of population tested in order to bring the apparent Reproduction number (Rt) below 1. Thus, it is important to consider non-uniform dynamics of viral spread and false negative rate in order to model effective testing plans.

Gradient Tracking by Yeast GPCRs in a Microfluidics Chamber.

Cells typically exist in a highly dynamic environment, which cannot easily be recreated in culture dishes or microwell plates. Microfluidic devices can provide precise control of the time, dose, and orientation of a stimulus, while simultaneously capturing quantitative single-cell data. The approach is particularly powerful when combined with the genetically tractable yeast model organism. The GPCR pathway in yeast is structurally conserved and functionally interchangeable with those in humans. We describe the implementation of a microfluidic device to investigate morphological and transcriptional responses of yeast to a gradient or pulse administration of a GPCR ligand, the peptide mating pheromone α-factor.

Karyopherin alpha7 (KPNA7), a divergent member of the importin alpha family of nuclear import receptors.

BACKGROUND: Classical nuclear localization signal (NLS) dependent nuclear import is carried out by a heterodimer of importin alpha and importin beta. NLS cargo is recognized by importin alpha, which is bound by importin beta. Importin beta mediates translocation of the complex through the central channel of the nuclear pore, and upon reaching the nucleus, RanGTP binding to importin beta triggers disassembly of the complex. To date, six importin alpha family members, encoded by separate genes, have been described in humans. RESULTS: We sequenced and characterized a seventh member of the importin alpha family of transport factors, karyopherin alpha 7 (KPNA7), which is most closely related to KPNA2. The domain of KPNA7 that binds Importin beta (IBB) is divergent, and shows stronger binding to importin beta than the IBB domains from of other importin alpha family members. With regard to NLS recognition, KPNA7 binds to the retinoblastoma (RB) NLS to a similar degree as KPNA2, but it fails to bind the SV40-NLS and the human nucleoplasmin (NPM) NLS. KPNA7 shows a predominantly nuclear distribution under steady state conditions, which contrasts with KPNA2 which is primarily cytoplasmic. CONCLUSION: KPNA7 is a novel importin alpha family member in humans that belongs to the importin alpha2 subfamily. KPNA7 shows different subcellular localization and NLS binding characteristics compared to other members of the importin alpha family. These properties suggest that KPNA7 could be specialized for interactions with select NLS-containing proteins, potentially impacting developmental regulation.

Ligand binding to the androgen receptor induces conformational changes that regulate phosphatase interactions.

We describe a mechanism for protein phosphatase 2A (PP2A) targeting to the androgen receptor (AR) and provide insight into the more general issue of kinase and phosphatase interactions with AR. Simian virus 40 (SV40) small t antigen (ST) binding to N-terminal HEAT repeats in the PP2A A subunit induces structural changes transduced to C-terminal HEAT repeats. This enables the C-terminal HEAT repeats in the PP2A A subunit, including HEAT repeat 13, to discriminate between androgen- and androgen antagonist-induced AR conformations. The PP2A-AR interaction was used to show that an AR mutant in prostate cancer cells (T877A) is activated by multiple ligands without acquiring the same conformation as that induced by androgen. The correlation between androgen binding to AR and increased phosphorylation of the activation function 1 (AF-1) region implies that changes in AR conformation or chaperone composition are causal to kinase access to phosphorylation sites. However, AF-1 phosphorylation sites are kinase accessible prior to androgen binding. This suggests that androgens can enhance the phosphorylation state of AR either by negatively regulating the ability of the ligand-binding domain to bind phosphatases or by inducing an AR conformation that is resistant to phosphatase action. SV40 ST subverts this mechanism by promoting the direct transfer of PP2A onto androgen-bound AR, resulting in multisite dephosphorylation.

Hyperosmotic stress signaling to the nucleus disrupts the Ran gradient and the production of RanGTP.

The RanGTP gradient depends on nucleocytoplasmic shuttling of Ran and its nucleotide exchange in the nucleus. Here we show that hyperosmotic stress signaling induced by sorbitol disrupts the Ran protein gradient and reduces the production of RanGTP. Ran gradient disruption is rapid and is followed by early (10-20 min) and late (30-60 min) phases of recovery. Results from SB203580 and siRNA experiments suggest the stress kinase p38 is important for Ran gradient recovery. NTF2 and Mog1, which are transport factors that regulate the nuclear localization of Ran, showed kinetics of delocalization and recovery similar to Ran. Microinjection of a nuclear localization signal reporter protein revealed that sorbitol stress decreases the rate of nuclear import. Sorbitol stress also slowed RCC1 mobility in the nucleus, which is predicted to reduce RCC1 dissociation from chromatin and RanGTP production. This was tested using a FRET biosensor that registers nuclear RanGTP levels, which were reduced in response to sorbitol stress. Although sorbitol alters nucleotide levels, we show that inverting the GTP/GDP ratio in cells is not sufficient to disrupt the Ran gradient. Thus, the Ran system is a target of hyperosmotic stress signaling, and cells use protein localization-based mechanisms as part of a rapid stress response.

Temporal Dynamics of Viral Load and False negative Rate Influence the Levels of Testing Necessary to Combat COVID19 Spread.

Colleges and other organizations are considering testing plans to return to operation as the COVID19 pandemic continues. Pre-symptomatic spread and high false negative rates for testing may make it difficult to stop viral spread. Here, we develop a stochastic agent-based model of COVID19 in a university sized population, considering the dynamics of both viral load and false negative rate of tests on the ability of testing to combat viral spread. Reported dynamics of SARS-CoV-2 can lead to an apparent false negative rate from ~17% to ~48%. Nonuniform distributions of viral load and false negative rate lead to higher requirements for frequency and fraction of population tested in order to bring the apparent Reproduction number (Rt) below 1. Thus, it is important to consider non-uniform dynamics of viral spread and false negative rate in order to model effective testing plans.