ß -actin is essential for structural integrity and physiological function of the retina.

Lack of non-muscle ß -actin gene (Actb) leads to early embryonic lethality in mice, however mice with ß - to γ -actin replacement develop normally and show no detectable phenotypes at young age. Here we investigated the effect of this replacement in the retina. During aging, these mice have accelerated de-generation of retinal structure and function, including elongated microvilli and defective mitochondria of retinal pigment epithelium (RPE), abnormally bulging photoreceptor outer segments (OS) accompanied by reduced transducin concentration and light sensitivity, and accumulation of autofluorescent microglia cells in the subretinal space between RPE and OS. These defects are accompanied by changes in the F-actin binding of several key actin interacting partners, including ezrin, myosin, talin, and vinculin known to play central roles in modulating actin cytoskeleton and cell adhesion and mediating the phagocytosis of OS. Our data show that ß -actin protein is essential for maintaining normal retinal structure and function.

Differential N-terminal processing of beta and gamma actin.

Cytoplasmic beta- and gamma-actin are ubiquitously expressed in every eukaryotic cell. They are encoded by different genes, but their amino acid sequences differ only by four conservative substitutions at the N-termini, making it difficult to dissect their individual regulation. Here, we analyzed actin from cultured cells and tissues by mass spectrometry and found that beta, unlike gamma actin, undergoes sequential removal of N-terminal Asp residues, leading to truncated actin species found in both F- and G-actin preparations. This processing affects up to ∼3% of beta actin in different cell types. We used CRISPR/Cas-9 in cultured cells to delete two candidate enzymes capable of mediating this type of processing. This deletion abolishes most of the beta actin N-terminal processing and results in changes in F-actin levels, cell spreading, filopodia formation, and cell migration. Our results demonstrate previously unknown isoform-specific actin regulation that can potentially affect actin functions in cells.

A new mechanism of fibronectin fibril assembly revealed by live imaging and super-resolution microscopy.

Fibronectin (Fn1) fibrils have long been viewed as continuous fibers composed of extended, periodically aligned Fn1 molecules. However, our live-imaging and single-molecule localization microscopy data are inconsistent with this traditional view and show that Fn1 fibrils are composed of roughly spherical nanodomains containing six to eleven Fn1 dimers. As they move toward the cell center, Fn1 nanodomains become organized into linear arrays, in which nanodomains are spaced with an average periodicity of 105±17 nm. Periodical Fn1 nanodomain arrays can be visualized between cells in culture and within tissues; they are resistant to deoxycholate treatment and retain nanodomain periodicity in the absence of cells. The nanodomain periodicity in fibrils remained constant when probed with antibodies recognizing distinct Fn1 epitopes or combinations of antibodies recognizing epitopes spanning the length of Fn1. Treatment with FUD, a peptide that binds the Fn1 N-terminus and disrupts Fn1 fibrillogenesis, blocked the organization of Fn1 nanodomains into periodical arrays. These studies establish a new paradigm of Fn1 fibrillogenesis. This article has an associated First Person interview with the first author of the paper.

Protein Posttranslational Signatures Identified in COVID-19 Patient Plasma.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a highly contagious virus of the coronavirus family that causes coronavirus disease-19 (COVID-19) in humans and a number of animal species. COVID-19 has rapidly propagated in the world in the past 2 years, causing a global pandemic. Here, we performed proteomic analysis of plasma samples from COVID-19 patients compared to healthy control donors in an exploratory study to gain insights into protein-level changes in the patients caused by SARS-CoV-2 infection and to identify potential proteomic and posttranslational signatures of this disease. Our results suggest a global change in protein processing and regulation that occurs in response to SARS-CoV-2, and the existence of a posttranslational COVID-19 signature that includes an elevation in threonine phosphorylation, a change in glycosylation, and a decrease in arginylation, an emerging posttranslational modification not previously implicated in infectious disease. This study provides a resource for COVID-19 researchers and, longer term, and will inform our understanding of this disease and its treatment.

Different translation dynamics of ß- and γ-actin regulates cell migration.

ß- and γ-cytoplasmic actins are ubiquitously expressed in every cell type and are nearly identical at the amino acid level but play vastly different roles in vivo. Their essential roles in embryogenesis and mesenchymal cell migration critically depend on the nucleotide sequences of their genes, rather than their amino acid sequences; however, it is unclear which gene elements underlie this effect. Here we address the specific role of the coding sequence in ß- and γ-cytoplasmic actins' intracellular functions, using stable polyclonal populations of immortalized mouse embryonic fibroblasts with exogenously expressed actin isoforms and their 'codon-switched' variants. When targeted to the cell periphery using ß-actin 3'UTR; ß-actin and γ-actin have differential effects on cell migration. These effects directly depend on the coding sequence. Single-molecule measurements of actin isoform translation, combined with fluorescence recovery after photobleaching, demonstrate a pronounced difference in ß- and γ-actins' translation elongation rates in cells, leading to changes in their dynamics at focal adhesions, impairments in actin bundle formation, and reduced cell anchoring to the substrate during migration. Our results demonstrate that coding sequence-mediated differences in actin translation play a key role in cell migration.

Most mammalian cells make both ß- and γ-actin, two proteins which shape the cell's internal skeleton and its ability to migrate. The molecules share over 99% of their sequence, yet they play distinct roles. In fact, deleting the ß-actin gene in mice causes death in the womb, while the animals can survive with comparatively milder issues without their γ-actin gene. How two similar proteins can have such different biological roles is a long-standing mystery. A closer look could hold some clues: ß- and γ-actin may contain the same blocks (or amino acids), but the genetic sequences that encode these proteins differ by about 13%. This is because different units of genetic information ­ known as synonymous codons ­ can encode the same amino acid. These 'silent substitutions' have no effect on the sequence of the proteins, yet a cell reads synonymous codons (and therefore produces proteins) at different speeds. To find out the impact of silent substitutions, Vedula et al. swapped the codons for the two proteins, forcing mouse cells to produce ß-actin using γ-actin codons, and vice versa. Cells with non-manipulated γ-actin and those with ß-actin made using γ-actin codons could move much faster than cells with ß-actin. This suggested that silent substitutions were indeed affecting the role of the protein. Vedula et al. found that cells read γ-codons ­ and therefore made γ-actin ­ much more slowly than ß-codons: this also affected how quickly the protein could be dispatched where it was needed in the cell. Slower production meant that bundles of γ-actin were shorter, which allowed cells to move faster by providing a weaker anchoring system. Overall, this work provides new links between silent substitutions and protein behavior, a relatively new research area which is likely to shed light on other protein families.

Arginylation Regulates G-protein Signaling in the Retina.

Arginylation is a post-translational modification mediated by the arginyltransferase (Ate1). We recently showed that conditional deletion of Ate1 in the nervous system leads to increased light-evoked response sensitivities of ON-bipolar cells in the retina, indicating that arginylation regulates the G-protein signaling complexes of those neurons and/or photoreceptors. However, none of the key players in the signaling pathway were previously shown to be arginylated. Here we show that Gαt1, Gß1, RGS6, and RGS7 are arginylated in the retina and RGS6 and RGS7 protein levels are elevated in Ate1 knockout, suggesting that arginylation plays a direct role in regulating their protein level and the G-protein-mediated responses in the retina.

The makings of the 'actin code': regulation of actin's biological function at the amino acid and nucleotide level.

The actin cytoskeleton plays key roles in every eukaryotic cell and is essential for cell adhesion, migration, mechanosensing, and contractility in muscle and non-muscle tissues. In higher vertebrates, from birds through to mammals, actin is represented by a family of six conserved genes. Although these genes have evolved independently for more than 100 million years, they encode proteins with ≥94% sequence identity, which are differentially expressed in different tissues, and tightly regulated throughout embryogenesis and adulthood. It has been previously suggested that the existence of such similar actin genes is a fail-safe mechanism to preserve the essential function of actin through redundancy. However, knockout studies in mice and other organisms demonstrate that the different actins have distinct biological roles. The mechanisms maintaining this distinction have been debated in the literature for decades. This Review summarizes data on the functional regulation of different actin isoforms, and the mechanisms that lead to their different biological roles in vivo We focus here on recent studies demonstrating that at least some actin functions are regulated beyond the amino acid level at the level of the actin nucleotide sequence.

Rapid and dynamic arginylation of the leading edge ß-actin is required for cell migration.

ß-actin plays key roles in cell migration. Our previous work demonstrated that ß-actin in migratory non-muscle cells is N-terminally arginylated and that this arginylation is required for normal lamellipodia extension. Here, we examined the function of ß-actin arginylation in cell migration. We found that arginylated ß-actin is concentrated at the leading edge of lamellipodia and that this enrichment is abolished after serum starvation as well as in contact-inhibited cells in confluent cultures, suggesting that arginylated ß-actin at the cell leading edge is coupled to active migration. Arginylated actin levels exhibit dynamic changes in response to cell stimuli, lowered after serum starvation and dramatically elevating within minutes after cell stimulation by readdition of serum or lysophosphatidic acid. These dynamic changes require active translation and are not seen in confluent contact-inhibited cell cultures. Microinjection of arginylated actin antibodies into cells severely and specifically inhibits their migration rates. Together, these data strongly suggest that arginylation of ß-actin is a tightly regulated dynamic process that occurs at the leading edge of locomoting cells in response to stimuli and is integral to the signaling network that regulates cell migration.

Diverse functions of homologous actin isoforms are defined by their nucleotide, rather than their amino acid sequence.

ß- and γ-cytoplasmic actin are nearly indistinguishable in their amino acid sequence, but are encoded by different genes that play non-redundant biological roles. The key determinants that drive their functional distinction are unknown. Here, we tested the hypothesis that ß- and γ-actin functions are defined by their nucleotide, rather than their amino acid sequence, using targeted editing of the mouse genome. Although previous studies have shown that disruption of ß-actin gene critically impacts cell migration and mouse embryogenesis, we demonstrate here that generation of a mouse lacking ß-actin protein by editing ß-actin gene to encode γ-actin protein, and vice versa, does not affect cell migration and/or organism survival. Our data suggest that the essential in vivo function of ß-actin is provided by the gene sequence independent of the encoded protein isoform. We propose that this regulation constitutes a global 'silent code' mechanism that controls the functional diversity of protein isoforms.

Arginyltransferase ATE1 is targeted to the neuronal growth cones and regulates neurite outgrowth during brain development.

Arginylation is an emerging protein modification mediated by arginyltransferase ATE1, shown to regulate embryogenesis and actin cytoskeleton, however its functions in different physiological systems are not well understood. Here we analyzed the role of ATE1 in brain development and neuronal growth by producing a conditional mouse knockout with Ate1 deletion in the nervous system driven by Nestin promoter (Nes-Ate1 mice). These mice were weaker than wild type, resulting in low postnatal survival rates, and had abnormalities in the brain that suggested defects in neuronal migration. Cultured Ate1 knockout neurons showed a reduction in the neurite outgrowth and the levels of doublecortin and F-actin in the growth cones. In wild type, ATE1 prominently localized to the growth cones, in addition to the cell bodies. Examination of the Ate1 mRNA sequence reveals the existence of putative zipcode-binding sequences involved in mRNA targeting to the cell periphery and local translation at the growth cones. Fluorescence in situ hybridization showed that Ate1 mRNA localized to the tips of the growth cones, likely due to zipcode-mediated targeting, and this localization coincided with spots of localization of arginylated ß-actin, which disappeared in the presence of protein synthesis inhibitors. We propose that zipcode-mediated co-targeting of Ate1 and ß-actin mRNA leads to localized co-translational arginylation of ß-actin that drives the growth cone migration and neurite outgrowth.

Quantifying cadherin mechanotransduction machinery assembly/disassembly dynamics using fluorescence covariance analysis.

Quantifying multi-molecular complex assembly in specific cytoplasmic compartments is crucial to understand how cells use assembly/disassembly of these complexes to control function. Currently, biophysical methods like Fluorescence Resonance Energy Transfer and Fluorescence Correlation Spectroscopy provide quantitative measurements of direct protein-protein interactions, while traditional biochemical approaches such as sub-cellular fractionation and immunoprecipitation remain the main approaches used to study multi-protein complex assembly/disassembly dynamics. In this article, we validate and quantify multi-protein adherens junction complex assembly in situ using light microscopy and Fluorescence Covariance Analysis. Utilizing specific fluorescently-labeled protein pairs, we quantified various stages of adherens junction complex assembly, the multiprotein complex regulating epithelial tissue structure and function following de novo cell-cell contact. We demonstrate: minimal cadherin-catenin complex assembly in the perinuclear cytoplasm and subsequent localization to the cell-cell contact zone, assembly of adherens junction complexes, acto-myosin tension-mediated anchoring, and adherens junction maturation following de novo cell-cell contact. Finally applying Fluorescence Covariance Analysis in live cells expressing fluorescently tagged adherens junction complex proteins, we also quantified adherens junction complex assembly dynamics during epithelial monolayer formation.

Balancing spatially regulated ß-actin translation and dynamin-mediated endocytosis is required to assemble functional epithelial monolayers.

Regulating adherens junction complex assembly/disassembly is critical to maintaining epithelial homeostasis in healthy epithelial tissues. Consequently, adherens junction structure and function is often perturbed in clinically advanced tumors of epithelial origin. Some of the most studied factors driving adherens junction complex perturbation in epithelial cancers are transcriptional and epigenetic down-regulation of E-cadherin expression. However, numerous reports demonstrate that post-translational regulatory mechanisms such as endocytosis also regulate early phases of epithelial-mesenchymal transition and metastatic progression. In already assembled healthy epithelia, E-cadherin endocytosis recycles cadherin-catenin complexes to regulate the number of mature adherens junctions found at cell-cell contact sites. However, following de novo epithelial cell-cell contact, endocytosis negatively regulates adherens junction assembly by removing E-cadherin from the cell surface. By contrast, following de novo epithelial cell-cell contact, spatially localized ß-actin translation drives cytoskeletal remodeling and consequently E-cadherin clustering at cell-cell contact sites and therefore positively regulates adherens junction assembly. In this report we demonstrate that dynamin-mediated endocytosis and ß-actin translation-dependent cadherin-catenin complex anchoring oppose each other following epithelial cell-cell contact. Consequently, the final extent of adherens junction assembly depends on which of these processes is dominant following epithelial cell-cell contact. We expressed ß-actin transcripts impaired in their ability to properly localize monomer synthesis (Δ3'UTR) in MDCK cells to perturb actin filament remodeling and anchoring, and demonstrate the resulting defect in adherens junction structure and function is rescued by inhibiting dynamin mediated endocytosis. Therefore, we demonstrate balancing spatially regulated ß-actin translation and dynamin-mediated endocytosis regulates epithelial monolayer structure and barrier function.

Rab8a vesicles regulate Wnt ligand delivery and Paneth cell maturation at the intestinal stem cell niche.

Communication between stem and niche supporting cells maintains the homeostasis of adult tissues. Wnt signaling is a crucial regulator of the stem cell niche, but the mechanism that governs Wnt ligand delivery in this compartment has not been fully investigated. We identified that Wnt secretion is partly dependent on Rab8a-mediated anterograde transport of Gpr177 (wntless), a Wnt-specific transmembrane transporter. Gpr177 binds to Rab8a, depletion of which compromises Gpr177 traffic, thereby weakening the secretion of multiple Wnts. Analyses of generic Wnt/ß-catenin targets in Rab8a knockout mouse intestinal crypts indicate reduced signaling activities; maturation of Paneth cells - a Wnt-dependent cell type - is severely affected. Rab8a knockout crypts show an expansion of Lgr5(+) and Hopx(+) cells in vivo. However, in vitro, the knockout enteroids exhibit significantly weakened growth that can be partly restored by exogenous Wnts or Gsk3ß inhibitors. Immunogold labeling and surface protein isolation identified decreased plasma membrane localization of Gpr177 in Rab8a knockout Paneth cells and fibroblasts. Upon stimulation by exogenous Wnts, Rab8a-deficient cells show ligand-induced Lrp6 phosphorylation and transcriptional reporter activation. Rab8a thus controls Wnt delivery in producing cells and is crucial for Paneth cell maturation. Our data highlight the profound tissue plasticity that occurs in response to stress induced by depletion of a stem cell niche signal.

CDC42 inhibition suppresses progression of incipient intestinal tumors.

Mutations in the APC or ß-catenin genes are well-established initiators of colorectal cancer, yet modifiers that facilitate the survival and progression of nascent tumor cells are not well defined. Using genetic and pharmacologic approaches in mouse colorectal cancer and human colorectal cancer xenograft models, we show that incipient intestinal tumor cells activate CDC42, an APC-interacting small GTPase, as a crucial step in malignant progression. In the mouse, Cdc42 ablation attenuated the tumorigenicity of mutant intestinal cells carrying single APC or ß-catenin mutations. Similarly, human colorectal cancer with relatively higher levels of CDC42 activity was particularly sensitive to CDC42 blockade. Mechanistic studies suggested that Cdc42 may be activated at different levels, including at the level of transcriptional activation of the stem cell-enriched Rho family exchange factor Arhgef4. Our results indicate that early-stage mutant intestinal epithelial cells must recruit the pleiotropic functions of Cdc42 for malignant progression, suggesting its relevance as a biomarker and therapeutic target for selective colorectal cancer intervention.

The ß-actin mRNA zipcode regulates epithelial adherens junction assembly but not maintenance.

Epithelial cell-cell contact stimulates actin cytoskeleton remodeling to down-regulate branched filament polymerization-driven lamellar protrusion and subsequently to assemble linear actin filaments required for E-cadherin anchoring during adherens junction complex assembly. In this manuscript, we demonstrate that de novo protein synthesis, the ß-actin 3' UTR, and the ß-actin mRNA zipcode are required for epithelial adherens junction complex assembly but not maintenance. Specifically, we demonstrate that perturbing cell-cell contact-localized ß-actin monomer synthesis causes epithelial adherens junction assembly defects. Consequently, inhibiting ß-actin mRNA zipcode/ZBP1 interactions with ß-actin mRNA zipcode antisense oligonucleotides, to intentionally delocalize ß-actin monomer synthesis, is sufficient to perturb adherens junction assembly following epithelial cell-cell contact. Additionally, we demonstrate active RhoA, the signal required to drive zipcode-mediated ß-actin mRNA targeting, is localized at epithelial cell-cell contact sites in a ß-actin mRNA zipcode-dependent manner. Moreover, chemically inhibiting Src kinase activity prevents the local stimulation of ß-actin monomer synthesis at cell-cell contact sites while inhibiting epithelial adherens junction assembly. Together, these data demonstrate that epithelial cell-cell contact stimulates ß-actin mRNA zipcode-mediated monomer synthesis to spatially regulate actin filament remodeling, thereby controlling adherens junction assembly to modulate cell and tissue adhesion.