Strategies for monitoring cell-cell interactions.

Multicellular organisms depend on physical cell-cell interactions to control physiological processes such as tissue formation, neurotransmission and immune response. These intercellular binding events can be both highly dynamic in their duration and complex in their composition, involving the participation of many different surface and intracellular biomolecules. Untangling the intricacy of these interactions and the signaling pathways they modulate has greatly improved insight into the biological processes that ensue upon cell-cell engagement and has led to the development of protein- and cell-based therapeutics. The importance of monitoring physical cell-cell interactions has inspired the development of several emerging approaches that effectively interrogate cell-cell interfaces with molecular-level detail. Specifically, the merging of chemistry- and biology-based technologies to deconstruct the complexity of cell-cell interactions has provided new avenues for understanding cell-cell interaction biology and opened opportunities for therapeutic development.

A Role for Basigin in Toxoplasma gondii Infection.

The role of specific host cell surface receptors during Toxoplasma gondii invasion of host cells is poorly defined. Here, we interrogated the role of the well-known malarial invasion receptor, basigin, in T. gondii infection of astrocytes. We found that primary astrocytes express two members of the BASIGIN (BSG) immunoglobulin family, basigin and embigin, but did not express neuroplastin. Antibody blockade of either basigin or embigin caused a significant reduction of parasite infectivity in astrocytes. The specific role of basigin during T. gondii invasion was further examined using a mouse astrocytic cell line (C8-D30), which exclusively expresses basigin. CRISPR-mediated deletion of basigin in C8-D30 cells resulted in decreased T. gondii infectivity. T. gondii replication and invasion efficiency were not altered by basigin deficiency, but parasite attachment to astrocytes was markedly reduced. We also conducted a proteomic screen to identify T. gondii proteins that interact with basigin. Toxoplasma-encoded cyclophilins, the protein 14-3-3, and protein disulfide isomerase (TgPDI) were among the putative basigin-ligands identified. Recombinant TgPDI produced in E. coli bound to basigin and pretreatment of tachyzoites with a PDI inhibitor decreased parasite attachment to host cells. Finally, mutagenesis of the active site cysteines of TgPDI abolished enzyme binding to basigin. Thus, basigin and its related immunoglobulin family members may represent host receptors that mediate attachment of T. gondii to diverse cell types.

Proteomic mapping of intercellular synaptic environments via flavin-dependent photoredox catalysis.

Receptor-ligand interactions play essential signaling roles within intercellular contact regions. This is particularly important within the context of the immune synapse where protein communication at the surface of physically interacting T cells and antigen-presenting cells regulate downstream immune signaling responses. To identify protein microenvironments within immunological synapses, we combined a flavin-dependent photocatalytic labeling strategy with quantitative mass spectrometry-based proteomics. Using α-PD-L1 or α-PD-1 single-domain antibody (VHH)-based photocatalyst targeting modalities, we profiled protein microenvironments within the intercellular region of an immune synapse-forming co-culture system. In addition to enrichment of both PD-L1 and PD-1 with either targeting modality, we also observed enrichment of both known immune synapse residing receptor-ligand pairs and surface proteins, as well as previously unknown synapse residing proteins.

The apical annuli of Toxoplasma gondii are composed of coiled-coil and signalling proteins embedded in the inner membrane complex sutures.

The apical annuli are among the most intriguing and understudied structures in the cytoskeleton of the apicomplexan parasite Toxoplasma gondii. We mapped the proteome of the annuli in Toxoplasma by reciprocal proximity biotinylation (BioID), and validated five apical annuli proteins (AAP1-5), Centrin2, and an apical annuli methyltransferase. Moreover, inner membrane complex (IMC) suture proteins connecting the alveolar vesicles were also detected and support annuli residence within the sutures. Super-resolution microscopy identified a concentric organisation comprising four rings with diameters ranging from 200 to 400 nm. The high prevalence of domain signatures shared with centrosomal proteins in the AAPs together with Centrin2 suggests that the annuli are related and/or derived from the centrosomes. Phylogenetic analysis revealed that the AAPs are conserved narrowly in coccidian, apicomplexan parasites that multiply by an internal budding mechanism. This suggests a role in replication, for example, to provide pores in the mother IMC permitting exchange of building blocks and waste products. However, presence of multiple signalling domains and proteins are suggestive of additional functions. Knockout of AAP4, the most conserved compound forming the largest ring-like structure, modestly decreased parasite fitness in vitro but had no significant impact on acute virulence in vivo. In conclusion, the apical annuli are composed of coiled-coil and signalling proteins assembled in a pore-like structure crossing the IMC barrier maintained during internal budding.

From structure to redox: The diverse functional roles of disulfides and implications in disease.

This review provides a comprehensive overview of the functional roles of disulfide bonds and their relevance to human disease. The critical roles of disulfide bonds in protein structure stabilization and redox regulation of protein activity are addressed. Disulfide bonds are essential to the structural stability of many proteins within the secretory pathway and can exist as intramolecular or inter-domain disulfides. The proper formation of these bonds often relies on folding chaperones and oxidases such as members of the protein disulfide isomerase (PDI) family. Many of the PDI family members catalyze disulfide-bond formation, reduction, and isomerization through redox-active disulfides and perturbed PDI activity is characteristic of carcinomas and neurodegenerative diseases.  In addition to catalytic function in oxidoreductases, redox-active disulfides are also found on a diverse array of cellular proteins and act to regulate protein activity and localization in response to oxidative changes in the local environment. These redox-active disulfides are either dynamic intramolecular protein disulfides or mixed disulfides with small-molecule thiols generating glutathionylation and cysteinylation adducts. The oxidation and reduction of redox-active disulfides are mediated by cellular reactive oxygen species and activity of reductases, such as glutaredoxin and thioredoxin.  Dysregulation of cellular redox conditions and resulting changes in mixed disulfide formation are directly linked to diseases such as cardiovascular disease and Parkinson's disease.

Transcriptomic analysis using RNA sequencing and phenotypic analysis of Salmonella enterica after acid exposure for different time durations using adaptive laboratory evolution.

Introduction: This study is the final part of a two-part series that delves into the molecular mechanisms driving adaptive laboratory evolution (ALE) of Salmonella enterica in acid stress. The phenotypic and transcriptomic alterations in the acid-evolved lineages (EL) of Salmonella enterica serovar Enteritidis after 70 days of acid stress exposure were analyzed. Materials and methods: The stability of phenotypic changes observed after 70 days in acetic acid was explored after stress removal using a newly developed evolutionary lineage EL5. Additionally, the impact of short-term acid stress on the previously adapted lineage EL4 was also examined. Results: The results indicate that the elevated antibiotic minimum inhibitory concentration (MIC) observed after exposure to acetic acid for 70 days was lost when acid stress was removed. This phenomenon was observed against human antibiotics such as meropenem, ciprofloxacin, gentamicin, and streptomycin. The MIC of meropenem in EL4 on day 70 was 0.094 mM, which dropped to 0.032 mM when removed from acetic acid stress after day 70. However, after stress reintroduction, the MIC swiftly elevated, and within 4 days, it returned to 0.094 mM. After 20 more days of adaptation in acetic acid, the meropenem MIC increased to 0.125 mM. The other human antibiotics that were tested exhibited a similar trend. The MIC of acetic acid in EL4 on day 70 was observed to be 35 mM, which remained constant even after the removal of acetic acid stress. Readaptation of EL4 in acetic acid for 20 more days caused the acetic acid MIC to increase to 37 mM. Bacterial whole genome sequencing of EL5 revealed base substitutions in several genes involved in pathogenesis, such as the phoQ and wzc genes. Transcriptomic analysis of EL5 revealed upregulation of virulence, drug resistance, toxin-antitoxin, and iron metabolism genes. Unstable Salmonella small colony variants (SSCV) of S. Enteritidis were also observed in EL5 as compared to the wild-type unevolved S. Enteritidis. Discussion: This study presents a comprehensive understanding of the evolution of the phenotypic, genomic, and transcriptomic changes in S. Enteritidis due to prolonged acid exposure through ALE.

Chemical mutagenesis of Listeria monocytogenes for increased tolerance to benzalkonium chloride shows independent genetic underpinnings and off-target antibiotic resistance.

Listeria monocytogenes, a potentially fatal foodborne pathogen commonly found in food processing facilities, creates a significant economic burden that totals more than $2 billion annually in the United States due to outbreaks. Quaternary ammonium compounds (QACs), including benzalkonium chloride (BAC), are among the most widely used sanitizers to inhibit the growth and spread of L. monocytogenes from food processing facilities. However, resistance to QACs has been increasing in L. monocytogenes and different genetic mechanisms conferring resistance have been discovered. Here, we used ethyl methanesulfonate (EMS) to chemically mutagenize the BAC-susceptible strain, L. monocytogenes FSL-N1-304. We isolated two mutants with increased tolerance to BAC compared to the parental strain. Next, we assessed the off-target effect of increased tolerance to BAC by measuring the minimum inhibitory concentrations (MICs) of a diverse set of antibiotics, revealing that mut-1 and mut-2 displayed significantly increased resistance to fluoroquinolone antibiotics compared to the parental strain. A hemolysis assay was then used to investigate a potential correlation between BAC tolerance and virulence. Interestingly, mut-1 and mut-2 both exhibited significantly higher hemolysis percentage than the parental strain. We then sequenced the genomes of the parental strain and both mutants to identify mutations that may be involved in the increased resistance to BAC. We identified 3 and 29 mutations in mut-1 and mut-2, respectively. mut-1 contained nonsynonymous mutations in dagK (a diacylglycerol kinase), lmo2768 (a permease-encoding gene), and lmo0186 (resuscitation promoting factor). mut-2 contained a nonsense mutation in the nucleotide excision repair enzyme UvrABC system protein B encoding gene, uvrB, which likely accounts for the higher number of mutations observed. Transcriptome analysis in the presence of BAC revealed that genes related to the phosphotransferase system and internalins were up-regulated in both mutants, suggesting their significance in the BAC stress response. These two mutants provide insights into alternative mechanisms for increased BAC tolerance and could further our understanding of how L. monocytogenes persists in the food processing environment.

Adaptive laboratory evolution of Salmonella enterica in acid stress.

Introduction: Adaptive laboratory evolution (ALE) studies play a crucial role in understanding the adaptation and evolution of different bacterial species. In this study, we have investigated the adaptation and evolution of Salmonella enterica serovar Enteritidis to acetic acid using ALE. Materials and methods: Acetic acid concentrations below the minimum inhibitory concentration (sub-MIC) were used. Four evolutionary lineages (EL), namely, EL1, EL2, EL3, and EL4, of S. Enteritidis were developed, each demonstrating varying levels of resistance to acetic acid. Results: The acetic acid MIC of EL1 remained constant at 27 mM throughout 70 days, while the MIC of EL2, EL3, and EL4 increased throughout the 70 days. EL4 was adapted to the highest concentration of acetic acid (30 mM) and demonstrated the highest increase in its MIC against acetic acid throughout the study, reaching an MIC of 35 mM on day 70. The growth rates of the evolved lineages increased over time and were dependent on the concentration of acetic acid used during the evolutionary process. EL4 had the greatest increase in growth rate, reaching 0.33 (h-1) after 70 days in the presence of 30 mM acetic acid as compared to EL1, which had a growth rate of 0.2 (h-1) after 70 days with no exposure to acetic acid. Long-term exposure to acetic acid led to an increased MIC of human antibiotics such as ciprofloxacin and meropenem against the S. enterica evolutionary lineages. The MIC of ciprofloxacin for EL1 stayed constant at 0.016 throughout the 70 days while that of EL4 increased to 0.047. Bacterial whole genome sequencing revealed single-nucleotide polymorphisms in the ELs in various genes known to be involved in S. enterica virulence, pathogenesis, and stress response including phoP, phoQ, and fhuA. We also observed genome deletions in some of the ELs as compared to the wild-type S. Enteritidis which may have contributed to the bacterial acid adaptation. Discussion: This study highlights the potential for bacterial adaptation and evolution under environmental stress and underscores the importance of understanding the development of cross resistance to antibiotics in S. enterica populations. This study serves to enhance our understanding of the pathogenicity and survival strategies of S. enterica under acetic acid stress.

High-resolution photocatalytic mapping of SARS-CoV-2 spike interactions on the cell surface.

Identifying virus-host interactions on the cell surface can improve our understanding of viral entry and pathogenesis. SARS-CoV-2, the causative agent of the COVID-19 disease, uses ACE2 as a receptor to enter cells. Yet the full repertoire of cell surface proteins that contribute to viral entry is unknown. We developed a photocatalyst-based viral-host protein microenvironment mapping platform (ViraMap) to probe the molecular neighborhood of the SARS-CoV-2 spike protein on the human cell surface. Application of ViraMap to ACE2-expressing cells captured ACE2, the established co-receptor NRP1, and several novel cell surface proteins. We systematically analyzed the relevance of these candidate proteins to SARS-CoV-2 entry by knockdown and overexpression approaches in pseudovirus and authentic infection models and identified PTGFRN and EFNB1 as bona fide viral entry factors. Our results highlight additional host targets that participate in SARS-CoV-2 infection and showcase ViraMap as a powerful platform for defining viral interactions on the cell surface.

Proteomic characterization of the Toxoplasma gondii cytokinesis machinery portrays an expanded hierarchy of its assembly and function.

The basal complex (BC) is essential for T. gondii cell division but mechanistic details are lacking. Here we report a reciprocal proximity based biotinylation approach to map the BC's proteome. We interrogate the resulting map for spatiotemporal dynamics and function by disrupting the expression of components. This highlights four architecturally distinct BC subcomplexes, the compositions of which change dynamically in correlation with changes in BC function. We identify BCC0 as a protein undergirding BC formation in five foci that precede the same symmetry seen in the apical annuli and IMC sutures. Notably, daughter budding from BCC0 progresses bidirectionally: the apical cap in apical and the rest of the IMC in basal direction. Furthermore, the essential role of the BC in cell division is contained in BCC4 and MORN1 that form a 'rubber band' to sequester the basal end of the assembling daughter cytoskeleton. Finally, we assign BCC1 to the non-essential, final BC constriction step.

Nanoscale Mapping of EGFR and c-MET Protein Environments on Lung Cancer Cell Surfaces via Therapeutic Antibody Photocatalyst Conjugates.

Receptor tyrosine kinases are involved in essential signaling roles that impact cell growth, differentiation, and proliferation. The overexpression or mutation of these proteins can lead to aberrant signaling that has been directly linked to a number of diseases including cancer cell formation and progression. This has led to intense clinical focus on modulating RTK activity through direct targeting of signaling activity or cell types harboring aberrant RTK behavior. In particular, epidermal growth factor receptor (EGFR) has attracted intense clinical attention due to the impact of inhibiting this RTK on tumor growth. However, mutations incurred through targeting EGFR have led to therapeutic resistance that involves not only direct mutations to the EGFR protein but also the involvement of other RTKs, such as c-MET, that can overcome therapeutic-based EGFR inhibition effects. This has, not surprisingly, led to co-targeting strategies of RTKs such as EGFR and c-MET to overcome resistance mechanisms. While the ability to co-target these proteins has led to success in the clinic, a more comprehensive understanding of their proximal environments, particularly in the context of therapeutic modalities, could further enhance both our understanding of their signaling biology and provide additional avenues for targeting these surface proteins. Thus, to investigate EGFR and c-MET protein microenvironments, we utilized our recently developed iridium photocatalyst-based microenvironment mapping technology to catalog EGFR and c-MET surface environments on non-small cell lung cancer cell lines. Through this approach, we enriched EGFR and c-MET from the cell surface and identified known EGFR and c-MET associators as well as previously unidentified proximal proteins.

Population Genomic Analysis of Listeria monocytogenes From Food Reveals Substrate-Specific Genome Variation.

Listeria monocytogenes is the major causative agent of the foodborne illness listeriosis. Listeriosis presents as flu-like symptoms in healthy individuals, and can be fatal for children, elderly, pregnant women, and immunocompromised individuals. Estimates suggest that L. monocytogenes results in ∼1,600 illnesses and ∼260 deaths annually in the United States. L. monocytogenes can survive and persist in a variety of harsh environments, including conditions encountered in production of fermented dairy products such as cheese. For instance, microbial growth is often limited in soft cheese fermentation because of harsh pH, water content, and salt concentrations. However, L. monocytogenes has caused a number of deadly listeriosis outbreaks through the contamination of cheese. The purpose of this study was to understand if genetically distinct populations of L. monocytogenes are associated with particular foods, including cheese and dairy. To address this goal, we analyzed the population genetic structure of 504 L. monocytogenes strains isolated from food with publicly available genome assemblies. We identified 10 genetically distinct populations spanning L. monocytogenes lineages 1, II, and III and serotypes 1/2a, 1/2b, 1/2c, 4b, and 4c. We observed an overrepresentation of isolates from specific populations with cheese (population 2), fruit/vegetable (population 2), seafood (populations 5, 8 and 9) and meat (population 10). We used the Large Scale Blast Score Ratio pipeline and Roary to identify genes unique to population 1 and population 2 in comparison with all other populations, and screened for the presence of antimicrobial resistance genes and virulence genes across all isolates. We identified > 40 genes that were present at high frequency in population 1 and population 2 and absent in most other isolates. Many of these genes encoded for transcription factors, and cell surface anchored proteins. Additionally, we found that the virulence genes aut and ami were entirely or partially deleted in population 2. These results indicate that some L. monocytogenes populations may exhibit associations with particular foods, including cheese, and that gene content may contribute to this pattern.

Characterization of the roles of activated charcoal and Chelex in the induction of PrfA regulon expression in complex medium.

The foodborne pathogen Listeria monocytogenes is able to survive across a wide range of intra- and extra-host environments by appropriately modulating gene expression patterns in response to different stimuli. Positive Regulatory Factor A (PrfA) is the major transcriptional regulator of virulence gene expression in L. monocytogenes. It has long been known that activated charcoal is required to induce the expression of PrfA-regulated genes in complex media, such as Brain Heart Infusion (BHI), but not in chemically defined media. In this study, we show that the expression of the PrfA-regulated hly, which encodes listeriolysin O, is induced 5- and 8-fold in L. monocytogenes cells grown in Chelex-treated BHI (Ch-BHI) and in the presence of activated charcoal (AC-BHI), respectively, relative to cells grown in BHI medium. Specifically, we show that metal ions present in BHI broth plays a role in the reduced expression of the PrfA regulon. In addition, we show that expression of hly is induced when the levels of bioavailable extra- or intercellular iron are reduced. L. monocytogenes cells grown Ch-BHI and AC-BHI media showed similar levels of resistance to the iron-activated antibiotic, streptonigrin, indicating that activated charcoal reduces the intracellular labile iron pool. Metal depletion and exogenously added glutathione contributed synergistically to PrfA-regulated gene expression since glutathione further increased hly expression in metal-depleted BHI but not in BHI medium. Analyses of transcriptional reporter fusion expression patterns revealed that genes in the PrfA regulon are differentially expressed in response to metal depletion, metal excess and exogenous glutathione. Our results suggest that metal ion abundance plays a role in modulating expression of PrfA-regulated virulence genes in L. monocytogenes.

Ferlins and TgDOC2 in Toxoplasma Microneme, Rhoptry and Dense Granule Secretion.

The host cell invasion process of apicomplexan parasites like Toxoplasma gondii is facilitated by sequential exocytosis of the microneme, rhoptry and dense granule organelles. Exocytosis is facilitated by a double C2 domain (DOC2) protein family. This class of C2 domains is derived from an ancestral calcium (Ca2+) binding archetype, although this feature is optional in extant C2 domains. DOC2 domains provide combinatorial power to the C2 domain, which is further enhanced in ferlins that harbor 5-7 C2 domains. Ca2+ conditionally engages the C2 domain with lipids, membranes, and/or proteins to facilitating vesicular trafficking and membrane fusion. The widely conserved T. gondii ferlins 1 (FER1) and 2 (FER2) are responsible for microneme and rhoptry exocytosis, respectively, whereas an unconventional TgDOC2 is essential for microneme exocytosis. The general role of ferlins in endolysosmal pathways is consistent with the repurposed apicomplexan endosomal pathways in lineage specific secretory organelles. Ferlins can facilitate membrane fusion without SNAREs, again pertinent to the Apicomplexa. How temporal raises in Ca2+ combined with spatiotemporally available membrane lipids and post-translational modifications mesh to facilitate sequential exocytosis events is discussed. In addition, new data on cross-talk between secretion events together with the identification of a new microneme protein, MIC21, is presented.

Profiling Cysteine Reactivity and Oxidation in the Endoplasmic Reticulum.

The endoplasmic reticulum (ER) is the initial site of biogenesis of secretory pathway proteins, including proteins localized to the ER, Golgi, lysosomes, intracellular vesicles, plasma membrane, and extracellular compartments. Proteins within the secretory pathway contain a high abundance of disulfide bonds to protect against the oxidative extracellular environment. These disulfide bonds are typically formed within the ER by a variety of oxidoreductases, including members of the protein disulfide isomerase (PDI) family. Here, we establish chemoproteomic platforms to identify oxidized and reduced cysteine residues within the ER. Subcellular fractionation methods were utilized to enrich for the ER and significantly enhance the coverage of ER-localized cysteine residues. Reactive-cysteine profiling ranked ∼900 secretory pathway cysteines by reactivity with an iodoacetamide-alkyne probe, revealing functional cysteines annotated to participate in disulfide bonds, or S-palmitoylation sites within proteins. Through application of a variation of the OxICAT protocol for quantifying cysteine oxidation, the percentages of oxidation for each of ∼700 ER-localized cysteines were calculated. Lastly, perturbation of ER function, through chemical induction of ER stress, was used to investigate the effect of initiation of the unfolded protein response (UPR) on ER-localized cysteine oxidation. Together, these studies establish a platform for identifying reactive and functional cysteine residues on proteins within the secretory pathway as well as for interrogating the effects of diverse cellular stresses on ER-localized cysteine oxidation.

Cysteine reactivity across the subcellular universe.

Cysteine residues are concentrated at key functional sites within proteins, performing diverse roles in metal binding, catalysis, and redox chemistry. Chemoproteomic platforms to interrogate the reactive cysteinome have developed significantly over the past 10 years, resulting in a greater understanding of cysteine functionality, modification, and druggability. Recently, chemoproteomic methods to examine reactive cysteine residues from specific subcellular organelles have provided significantly improved proteome coverage and highlights the unique functionalities of cysteine residues mediated by cellular localization. Here, the diverse physicochemical properties of the mammalian subcellular organelles are explored in the context of their effects on cysteine reactivity. The unique functions of cysteine residues found in the mitochondria and endoplasmic reticulum are highlighted, together with an overview into chemoproteomic platforms employed to investigate cysteine reactivity in subcellular organelles.

Citrullination of NF-κB p65 promotes its nuclear localization and TLR-induced expression of IL-1ß and TNFα.

Many citrullinated proteins are known autoantigens in rheumatoid arthritis, a disease mediated by inflammatory cytokines, such as tumor necrosis factor-α (TNFα). Citrullinated proteins are generated by converting peptidylarginine to peptidylcitrulline, a process catalyzed by the peptidylarginine deiminases (PADs), including PAD1 to PAD4 and PAD6. Several major risk factors for rheumatoid arthritis are associated with heightened citrullination. However, the physiological role of citrullination in immune cells is poorly understood. We report that suppression of PAD activity attenuates Toll-like receptor-induced expression of interleukin-1ß (IL-1ß) and TNFα by neutrophils in vivo and in vitro but not their global transcription activity. Mechanistically, PAD4 directly citrullinates nuclear factor κB (NF-κB) p65 and enhances the interaction of p65 with importin α3, which brings p65 into the nucleus. The citrullination-enhanced interaction of p65 with importin α3 and its nuclear translocation and transcriptional activity can be attributed to citrullination of four arginine residues located in the Rel homology domain of p65. Furthermore, a rheumatoid arthritis-prone variant of PAD4, carrying three missense mutations, is more efficient in interacting with p65 and enhancing NF-κB activity. Together, these data not only demonstrate a critical role of citrullination in an NF-κB-dependent expression of IL-1ß and TNFα but also provide a molecular mechanism by which heightened citrullination propagates inflammation in rheumatoid arthritis. Accordingly, attenuating p65-mediated production of IL-1ß and TNFα by blocking the citrullination of p65 has great therapeutic potential in rheumatoid arthritis.

A real-time, fluorescence-based assay for Rho-associated protein kinase activity.

Inhibitors of Rho-associated protein kinase (ROCK) enzymatic activity have been shown to reduce the invasive phenotype observed in metastatic hepatocellular carcinoma (HCC). We describe the design, synthesis, and evaluation of a direct probe for ROCK activity utilizing a phosphorylation-sensitive sulfonamido-oxine fluorophore, termed Sox. The Sox fluorophore undergoes an increase in fluorescence upon phosphorylation of a proximal amino acid via chelation-enhanced fluorescence (CHEF, ex. = 360 nm and em. = 485 nm), allowing for the direct visualization of the rate of phosphate addition to a peptide substrate over time. Our optimal probe design, ROCK-S1, is capable of sensitively reporting ROCK activity with a limit of detection of 10 pM and a high degree of reproducibility (Z'-factor = 0.6 at 100 pM ROCK2). As a proof-of-principle for high-throughput screening (HTS) we demonstrate the ability to rapidly assess the efficacy of a 78 member, small molecule library against ROCK2 using a robotics platform. We identify two previously unreported ROCK2 inhibitor scaffolds, PHA665752 and IKK16, with IC50 values of 3.6 µM and 247 nM respectively. Lastly, we define conditions for selectively monitoring ROCK activity in the presence of potential off-target enzymes (PKCα, PKA, and PAK) with similar substrate specificities.