Dynamic states of eIF6 and SDS variants modulate interactions with uL14 of the 60S ribosomal subunit.

Assembly of ribosomal subunits into active ribosomal complexes is integral to protein synthesis. Release of eIF6 from the 60S ribosomal subunit primes 60S to associate with the 40S subunit and engage in translation. The dynamics of eIF6 interaction with the uL14 (RPL23) interface of 60S and its perturbation by somatic mutations acquired in Shwachman-Diamond Syndrome (SDS) is yet to be clearly understood. Here, by using a modified strategy to obtain high yields of recombinant human eIF6 we have uncovered the critical interface entailing eight key residues in the C-tail of uL14 that is essential for physical interactions between 60S and eIF6. Disruption of the complementary binding interface by conformational changes in eIF6 disease variants provide a mechanism for weakened interactions of variants with the 60S. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) analyses uncovered dynamic configurational rearrangements in eIF6 induced by binding to uL14 and exposed an allosteric interface regulated by the C-tail of eIF6. Disrupting key residues in the eIF6-60S binding interface markedly limits proliferation of cancer cells, which highlights the significance of therapeutically targeting this interface. Establishing these key interfaces thus provide a therapeutic framework for targeting eIF6 in cancers and SDS.

Anti-CRISPR proteins function through thermodynamic tuning and allosteric regulation of CRISPR RNA-guided surveillance complex.

CRISPR RNA-guided detection and degradation of foreign DNA is a dynamic process. Viruses can interfere with this cellular defense by expressing small proteins called anti-CRISPRs. While structural models of anti-CRISPRs bound to their target complex provide static snapshots that inform mechanism, the dynamics and thermodynamics of these interactions are often overlooked. Here, we use hydrogen deuterium exchange-mass spectrometry (HDX-MS) and differential scanning fluorimetry (DSF) experiments to determine how anti-CRISPR binding impacts the conformational landscape of the type IF CRISPR RNA guided surveillance complex (Csy) upon binding of two different anti-CRISPR proteins (AcrIF9 and AcrIF2). The results demonstrate that AcrIF2 binding relies on enthalpic stabilization, whereas AcrIF9 uses an entropy driven reaction to bind the CRISPR RNA-guided surveillance complex. Collectively, this work reveals the thermodynamic basis and mechanistic versatility of anti-CRISPR-mediated immune suppression. More broadly, this work presents a striking example of how allosteric effectors are employed to regulate nucleoprotein complexes.

Aerobic bacterial methane synthesis.

Reports of biogenic methane (CH4) synthesis associated with a range of organisms have steadily accumulated in the literature. This has not happened without controversy and in most cases the process is poorly understood at the gene and enzyme levels. In marine and freshwater environments, CH4 supersaturation of oxic surface waters has been termed the "methane paradox" because biological CH4 synthesis is viewed to be a strictly anaerobic process carried out by O2-sensitive methanogens. Interest in this phenomenon has surged within the past decade because of the importance of understanding sources and sinks of this potent greenhouse gas. In our work on Yellowstone Lake in Yellowstone National Park, we demonstrate microbiological conversion of methylamine to CH4 and isolate and characterize an Acidovorax sp. capable of this activity. Furthermore, we identify and clone a gene critical to this process (encodes pyridoxylamine phosphate-dependent aspartate aminotransferase) and demonstrate that this property can be transferred to Escherichia coli with this gene and will occur as a purified enzyme. This previously unrecognized process sheds light on environmental cycling of CH4, suggesting that O2-insensitive, ecologically relevant aerobic CH4 synthesis is likely of widespread distribution in the environment and should be considered in CH4 modeling efforts.

Core Protein-Directed Antivirals and Importin ß Can Synergistically Disrupt Hepatitis B Virus Capsids.

Viral structural proteins can have multiple activities. Antivirals that target structural proteins have potential to exhibit multiple antiviral mechanisms. Hepatitis B virus (HBV) core protein (Cp) is involved in most stages of the viral life cycle; it assembles into capsids, packages viral RNA, is a metabolic compartment for reverse transcription, interacts with nuclear trafficking machinery, and disassembles to release the viral genome into the nucleus. During nuclear localization, HBV capsids bind to host importins (e.g., Impß) via Cp's C-terminal domain (CTD); the CTD is localized to the interior of the capsid and is transiently exposed on the exterior. We used HAP12 as a representative Cp allosteric modulator (CpAM), a class of antivirals that inappropriately stimulates and misdirects HBV assembly and deforms capsids. CpAM impact on other aspects of the HBV life cycle is poorly understood. We investigate how HAP12 influences the interactions between empty or RNA-filled capsids with Impß and trypsin in vitro. We show that HAP12 can modulate CTD accessibility and capsid stability, depending on the saturation of HAP12-binding sites. We demonstrate that Impß synergistically contributes to capsid disruption at high levels of HAP12 saturation, using electron microscopy to visualize the disruption and rearrangement of Cp dimers into aberrant complexes. However, RNA-filled capsids resist the destabilizing effects of HAP12 and Impß. In summary, we show host protein-induced catalysis of capsid disruption, an unexpected additional mechanism of action for CpAMs. Potentially, untimely capsid disassembly can hamper the HBV life cycle and also cause the virus to become vulnerable to host innate immune responses. IMPORTANCE The HBV core, an icosahedral complex of 120 copies of the homodimeric core (capsid) protein with or without packaged nucleic acid, is transported to the host nucleus by its interaction with host importin proteins. Importin-core interaction requires the core protein C-terminal domain, which is inside the capsid, to "flip" to the capsid exterior. Core protein-directed drugs that affect capsid assembly and stability have been developed recently. We show that these molecules can, synergistically with importins, disrupt capsids. This mechanism of action, synergism with host protein, has the potential to disrupt the virus life cycle and activate the innate immune system.

Hydrogen-deuterium exchange reveals a dynamic DNA-binding map of replication protein A.

Replication protein A (RPA) binds to single-stranded DNA (ssDNA) and interacts with over three dozen enzymes and serves as a recruitment hub to coordinate most DNA metabolic processes. RPA binds ssDNA utilizing multiple oligosaccharide/oligonucleotide binding domains and based on their individual DNA binding affinities are classified as high versus low-affinity DNA-binding domains (DBDs). However, recent evidence suggests that the DNA-binding dynamics of DBDs better define their roles. Utilizing hydrogen-deuterium exchange mass spectrometry (HDX-MS), we assessed the ssDNA-driven dynamics of the individual domains of human RPA. As expected, ssDNA binding shows HDX changes in DBDs A, B, C, D and E. However, DBD-A and DBD-B are dynamic and do not show robust DNA-dependent protection. DBD-C displays the most extensive changes in HDX, suggesting a major role in stabilizing RPA on ssDNA. Slower allosteric changes transpire in the protein-protein interaction domains and linker regions, and thus do not directly interact with ssDNA. Within a dynamics-based model for RPA, we propose that DBD-A and -B act as the dynamic half and DBD-C, -D and -E function as the less-dynamic half. Thus, segments of ssDNA buried under the dynamic half are likely more readily accessible to RPA-interacting proteins.

Relationships Between Perceived Importance of Chaplain Presence and Health Professionals' Emotional Well-Being in the United States.

Hospital-based chaplains receive specialized training to provide spiritual support to patients and healthcare staff during difficult health transitions. However, the impact of perceived chaplain importance on healthcare staff's emotional and professional well-being is unclear. Healthcare staff (n = 1471) caring for patients in an acute care setting within a large health system answered demographic and emotional health questions in Research Electronic Data Capture (REDCap). Findings suggest that as perceived levels of chaplain importance increase, burnout may decrease and compassion satisfaction may improve. Chaplain presence in the hospital setting may support healthcare staff emotional and professional well-being following occupational stressors including COVID-19-related surges.

The flexible N-terminus of BchL autoinhibits activity through interaction with its [4Fe-4S] cluster and released upon ATP binding.

A key step in bacteriochlorophyll biosynthesis is the reduction of protochlorophyllide to chlorophyllide, catalyzed by dark-operative protochlorophyllide oxidoreductase. Dark-operative protochlorophyllide oxidoreductase contains two [4Fe-4S]-containing component proteins (BchL and BchNB) that assemble upon ATP binding to BchL to coordinate electron transfer and protochlorophyllide reduction. But the precise nature of the ATP-induced conformational changes is poorly understood. We present a crystal structure of BchL in the nucleotide-free form where a conserved, flexible region in the N-terminus masks the [4Fe-4S] cluster at the docking interface between BchL and BchNB. Amino acid substitutions in this region produce a hyperactive enzyme complex, suggesting a role for the N-terminus in autoinhibition. Hydrogen-deuterium exchange mass spectrometry shows that ATP binding to BchL produces specific conformational changes leading to release of the flexible N-terminus from the docking interface. The release also promotes changes within the local environment surrounding the [4Fe-4S] cluster and promotes BchL-complex formation with BchNB. A key patch of amino acids, Asp-Phe-Asp (the 'DFD patch'), situated at the mouth of the BchL ATP-binding pocket promotes intersubunit cross stabilization of the two subunits. A linked BchL dimer with one defective ATP-binding site does not support protochlorophyllide reduction, illustrating nucleotide binding to both subunits as a prerequisite for the intersubunit cross stabilization. The masking of the [4Fe-4S] cluster by the flexible N-terminal region and the associated inhibition of the activity is a novel mechanism of regulation in metalloproteins. Such mechanisms are possibly an adaptation to the anaerobic nature of eubacterial cells with poor tolerance for oxygen.

ROS-mediated PI3K activation drives mitochondrial transfer from stromal cells to hematopoietic stem cells in response to infection.

Hematopoietic stem cells (HSCs) undergo rapid expansion in response to stress stimuli. Here we investigate the bioenergetic processes which facilitate the HSC expansion in response to infection. We find that infection by Gram-negative bacteria drives an increase in mitochondrial mass in mammalian HSCs, which results in a metabolic transition from glycolysis toward oxidative phosphorylation. The initial increase in mitochondrial mass occurs as a result of mitochondrial transfer from the bone marrow stromal cells (BMSCs) to HSCs through a reactive oxygen species (ROS)-dependent mechanism. Mechanistically, ROS-induced oxidative stress regulates the opening of connexin channels in a system mediated by phosphoinositide 3-kinase (PI3K) activation, which allows the mitochondria to transfer from BMSCs into HSCs. Moreover, mitochondria transfer from BMSCs into HSCs, in the response to bacterial infection, occurs before the HSCs activate their own transcriptional program for mitochondrial biogenesis. Our discovery demonstrates that mitochondrial transfer from the bone marrow microenvironment to HSCs is an early physiologic event in the mammalian response to acute bacterial infection and results in bioenergetic changes which underpin emergency granulopoiesis.

Biophysical characterization of SARAH domain-mediated multimerization of Hippo pathway complexes in Drosophila.

Hippo pathway signaling limits cell growth and proliferation and maintains the stem-cell niche. These cellular events result from the coordinated activity of a core kinase cassette that is regulated, in part, by interactions involving Hippo, Salvador, and dRassF. These interactions are mediated by a conserved coiled-coil domain, termed SARAH, in each of these proteins. SARAH domain-mediated homodimerization of Hippo kinase leads to autophosphorylation and activation. Paradoxically, SARAH domain-mediated heterodimerization between Hippo and Salvador enhances Hippo kinase activity in cells, whereas complex formation with dRassF inhibits it. To better understand the mechanism by which each complex distinctly modulates Hippo kinase and pathway activity, here we biophysically characterized the entire suite of SARAH domain-mediated complexes. We purified the three SARAH domains from Drosophila melanogaster and performed an unbiased pulldown assay to identify all possible interactions, revealing that isolated SARAH domains are sufficient to recapitulate the cellular assemblies and that Hippo is a universal binding partner. Additionally, we found that the Salvador SARAH domain homodimerizes and demonstrate that this interaction is conserved in Salvador's mammalian homolog. Using native MS, we show that each of these complexes is dimeric in solution. We also measured the stability of each SARAH domain complex, finding that despite similarities at both the sequence and structural levels, SARAH domain complexes differ in stability. The identity, stoichiometry, and stability of these interactions characterized here comprehensively reveal the nature of SARAH domain-mediated complex formation and provide mechanistic insights into how SARAH domain-mediated interactions influence Hippo pathway activity.

Intestinal Microbiome-Macrophage Crosstalk Contributes to Cholestatic Liver Disease by Promoting Intestinal Permeability in Mice.

BACKGROUND AND AIMS: Mounting evidence supports an association between cholestatic liver disease and changes in the composition of the microbiome. Still, the role of the microbiome in the pathogenesis of this condition remains largely undefined. APPROACH AND RESULTS: To address this, we have used two experimental models, administering alpha-naphtylisocyanate or feeding a 0.1% 3,5-diethoxycarbonyl-1,4-dihydrocollidine diet, to induce cholestatic liver disease in germ-free mice and germ-free mice conventionalized with the microbiome from wild-type, specific pathogen-free animals. Next, we have inhibited macrophage activation by depleting these cells using clodronate liposomes and inhibiting the inflammasome with a specific inhibitor of NOD-, LRR-, and pyrin domain-containing protein 3. Our results demonstrate that cholestasis, the accumulation of bile acids in the liver, fails to promote liver injury in the absence of the microbiome in vivo. Additional in vitro studies supported that endotoxin sensitizes hepatocytes to bile-acid-induced cell death. We also demonstrate that during cholestasis, macrophages contribute to promoting intestinal permeability and to altered microbiome composition through activation of the inflammasome, overall leading to increased endotoxin flux into the cholestatic liver. CONCLUSIONS: We demonstrate that the intestinal microbiome contributes to cholestasis-mediated cell death and inflammation through mechanisms involving activation of the inflammasome in macrophages.

Health-Related Goal Setting and Achievement Among Veterans with High Technology Adoption.

BACKGROUND: There is increasing recognition of the importance of supporting patients in their health-related goals. Patient-provider discussions and health-related mobile applications (apps) can support patients to pursue health goals; however, their impact on patient goal setting and achievement is not well understood. OBJECTIVE: To examine the relationships between the following: (1) patient demographics, patient-provider discussions, and health-related goal setting and achievement, and (2) patient mobile health app use and goal achievement. DESIGN: Cross-sectional survey. PARTICIPANTS: Veterans who receive Veterans Health Administration (VA) healthcare and are users of VA patient-facing technology. MAIN MEASURES: Veteran demographics, goal-related behaviors, and goal achievement. METHODS: Veterans were invited to participate in a telephone survey. VA administrative data were linked to survey data for additional health and demographic information. Logistic regression models were run to identify factors that predict health-related goal setting and achievement. KEY RESULTS: Among respondents (n=2552), 75% of patients indicated having set health goals in the preceding 6 months and approximately 42% reported achieving their goal. Men (vs. women) had lower odds of setting goals (OR: 0.71; CI95: 0.53-0.97), as did individuals with worse (vs. better) health (OR: 0.18; CI95: 0.04-0.88). Individuals with advanced education-some college/college degrees, and post-college degrees (vs. no college education)-demonstrated higher odds of setting goals (OR: 1.35; CI95: 1.01-1.79; OR: 1.71; CI95: 1.28-2.28, respectively). Those who reported having discussed their goals with their providers were more likely to set goals (OR: 3.60; CI95: 2.97-4.35). Patient mobile health app use was not statistically associated with goal achievement. CONCLUSIONS: Efforts to further promote patient-led goal setting should leverage the influence of patient-provider conversations. Use of patient-facing technologies, specifically mobile health apps, may facilitate goal-oriented care, but further work is needed to examine the potential benefits of apps to support patient goals, particularly if providers discuss and endorse use of those apps with patients.

Facilitators and barriers to post-discharge pain assessment and triage: a qualitative study of nurses' and patients' perspectives.

BACKGROUND: After hospital discharge, patients can experience symptoms prompting them to seek acute medical attention. Early evaluation of patients' post-discharge symptoms by healthcare providers may improve appropriate healthcare utilization and patient safety. Post-discharge follow-up phone calls, which are used for routine transitional care in U.S. hospitals, serve as an important channel for provider-patient communication about symptoms. This study aimed to assess the facilitators and barriers to evaluating and triaging pain symptoms in cardiovascular patients through follow-up phone calls after their discharge from a large healthcare system in Central Massachusetts. We also discuss strategies that may help address the identified barriers. METHODS: Guided by the Practical, Robust, Implementation and Sustainability Model (PRISM), we completed semi-structured interviews with 7 nurses and 16 patients in 2020. Selected nurses conducted (or supervised) post-discharge follow-up calls on behalf of 5 clinical teams (2 primary care; 3 cardiology). We used thematic analysis to identify themes from interviews and mapped them to the domains of the PRISM model. RESULTS: Participants described common facilitators and barriers related to the four domains of PRISM: Intervention (I), Recipients (R), Implementation and Sustainability Infrastructure (ISI), and External Environment (EE). Facilitators include: (1) patients being willing to receive provider follow-up (R); (2) nurses experienced in symptom assessment (R); (3) good care coordination within individual clinical teams (R); (4) electronic health record system and call templates to support follow-up calls (ISI); and (5) national and institutional policies to support post-discharge follow-up (EE). Barriers include: (1) limitations of conducting symptom assessment by provider-initiated follow-up calls (I); (2) difficulty connecting patients and providers in a timely manner (R); (3) suboptimal coordination for transitional care among primary care and cardiology providers (R); and (4) lack of emphasis on post-discharge follow-up call reimbursement among cardiology clinics (EE). Specific barriers for pain assessment include: (1) concerns with pain medication misuse (R); and (2) no standardized pain assessment and triage protocol (ISI). CONCLUSIONS: Strategies to empower patients, facilitate timely patient-provider communication, and support care coordination regarding pain evaluation and treatment may reduce the barriers and improve processes and outcomes of pain assessment and triage.

The catalytic mechanism of electron-bifurcating electron transfer flavoproteins (ETFs) involves an intermediary complex with NAD.

Electron bifurcation plays a key role in anaerobic energy metabolism, but it is a relatively new discovery, and only limited mechanistic information is available on the diverse enzymes that employ it. Herein, we focused on the bifurcating electron transfer flavoprotein (ETF) from the hyperthermophilic archaeon Pyrobaculum aerophilum The EtfABCX enzyme complex couples NADH oxidation to the endergonic reduction of ferredoxin and exergonic reduction of menaquinone. We developed a model for the enzyme structure by using nondenaturing MS, cross-linking, and homology modeling in which EtfA, -B, and -C each contained FAD, whereas EtfX contained two [4Fe-4S] clusters. On the basis of analyses using transient absorption, EPR, and optical titrations with NADH or inorganic reductants with and without NAD+, we propose a catalytic cycle involving formation of an intermediary NAD+-bound complex. A charge transfer signal revealed an intriguing interplay of flavin semiquinones and a protein conformational change that gated electron transfer between the low- and high-potential pathways. We found that despite a common bifurcating flavin site, the proposed EtfABCX catalytic cycle is distinct from that of the genetically unrelated bifurcating NADH-dependent ferredoxin NADP+ oxidoreductase (NfnI). The two enzymes particularly differed in the role of NAD+, the resting and bifurcating-ready states of the enzymes, how electron flow is gated, and the two two-electron cycles constituting the overall four-electron reaction. We conclude that P. aerophilum EtfABCX provides a model catalytic mechanism that builds on and extends previous studies of related bifurcating ETFs and can be applied to the large bifurcating ETF family.

Fine-Tuning of Sirtuin 1 Expression Is Essential to Protect the Liver From Cholestatic Liver Disease.

Cholestasis comprises aetiologically heterogeneous conditions characterized by accumulation of bile acids in the liver that actively contribute to liver damage. Sirtuin 1 (SIRT1) regulates liver regeneration and bile acid metabolism by modulating farnesoid X receptor (FXR); we here investigate its role in cholestatic liver disease. We determined SIRT1 expression in livers from patients with cholestatic disease, in two experimental models of cholestasis, as well as in human and murine liver cells in response to bile acid loading. SIRT1-overexpressing (SIRToe ) and hepatocyte-specific SIRT1-KO (knockout) mice (SIRThep-/- ) were subjected to bile duct ligation (BDL) and were fed with a 0.1% DDC (3,5-diethoxycarbonyl-1,4-dihydrocollidine) diet to determine the biological relevance of SIRT1 during cholestasis. The effect of NorUDCA (24-norursodeoxycholic acid) was tested in BDL/SIRToe mice. We found that SIRT1 was highly expressed in livers from cholestatic patients, mice after BDL, and Mdr2 knockout mice (Mdr2-/- ) animals. The detrimental effects of SIRT1 during cholestasis were validated in vivo and in vitro. SIRToe mice showed exacerbated parenchymal injury whereas SIRThep-/- mice evidenced a moderate improvement after BDL and 0.1% DDC feeding. Likewise, hepatocytes isolated from SIRToe mice showed increased apoptosis in response to bile acids, whereas a significant reduction was observed in SIRThep-/- hepatocytes. Importantly, the decrease, but not complete inhibition, of SIRT1 exerted by norUDCA treatment correlated with pronounced improvement in liver parenchyma in BDL/SIRToe mice. Interestingly, both SIRT1 overexpression and hepatocyte-specific SIRT1 depletion correlated with inhibition of FXR, whereas modulation of SIRT1 by NorUDCA associated with restored FXR signaling. Conclusion: SIRT1 expression is increased during human and murine cholestasis. Fine-tuning expression of SIRT1 is essential to protect the liver from cholestatic liver damage.

Salvador has an extended SARAH domain that mediates binding to Hippo kinase.

The Hippo pathway controls cell proliferation and differentiation through the precisely tuned activity of a core kinase cassette. The activity of Hippo kinase is modulated by interactions between its C-terminal coiled-coil, termed the SARAH domain, and the SARAH domains of either dRassF or Salvador. Here, we wanted to understand the molecular basis of SARAH domain-mediated interactions and their influence on Hippo kinase activity. We focused on Salvador, a positive effector of Hippo activity and the least well-characterized SARAH domain-containing protein. We determined the crystal structure of a complex between Salvador and Hippo SARAH domains from Drosophila This structure provided insight into the organization of the Salvador SARAH domain including a folded N-terminal extension that expands the binding interface with Hippo SARAH domain. We also found that this extension improves the solubility of the Salvador SARAH domain, enhances binding to Hippo, and is unique to Salvador. We therefore suggest expanding the definition of the Salvador SARAH domain to include this extended region. The heterodimeric assembly observed in the crystal was confirmed by cross-linked MS and provided a structural basis for the mutually exclusive interactions of Hippo with either dRassF or Salvador. Of note, Salvador influenced the kinase activity of Mst2, the mammalian Hippo homolog. In co-transfected HEK293T cells, human Salvador increased the levels of Mst2 autophosphorylation and Mst2-mediated phosphorylation of select substrates, whereas Salvador SARAH domain inhibited Mst2 autophosphorylation in vitro These results suggest Salvador enhances the effects of Hippo kinase activity at multiple points in the Hippo pathway.

The Electron Bifurcating FixABCX Protein Complex from Azotobacter vinelandii: Generation of Low-Potential Reducing Equivalents for Nitrogenase Catalysis.

The biological reduction of dinitrogen (N2) to ammonia (NH3) by nitrogenase is an energetically demanding reaction that requires low-potential electrons and ATP; however, pathways used to deliver the electrons from central metabolism to the reductants of nitrogenase, ferredoxin or flavodoxin, remain unknown for many diazotrophic microbes. The FixABCX protein complex has been proposed to reduce flavodoxin or ferredoxin using NADH as the electron donor in a process known as electron bifurcation. Herein, the FixABCX complex from Azotobacter vinelandii was purified and demonstrated to catalyze an electron bifurcation reaction: oxidation of NADH (Em = -320 mV) coupled to reduction of flavodoxin semiquinone (Em = -460 mV) and reduction of coenzyme Q (Em = 10 mV). Knocking out fix genes rendered Δrnf A. vinelandii cells unable to fix dinitrogen, confirming that the FixABCX system provides another route for delivery of electrons to nitrogenase. Characterization of the purified FixABCX complex revealed the presence of flavin and iron-sulfur cofactors confirmed by native mass spectrometry, electron paramagnetic resonance spectroscopy, and transient absorption spectroscopy. Transient absorption spectroscopy further established the presence of a short-lived flavin semiquinone radical, suggesting that a thermodynamically unstable flavin semiquinone may participate as an intermediate in the transfer of an electron to flavodoxin. A structural model of FixABCX, generated using chemical cross-linking in conjunction with homology modeling, revealed plausible electron transfer pathways to both high- and low-potential acceptors. Overall, this study informs a mechanism for electron bifurcation, offering insight into a unique method for delivery of low-potential electrons required for energy-intensive biochemical conversions.

A chemokine self-presentation mechanism involving formation of endothelial surface microstructures.

Endothelial surface microstructures have been described previously under inflammatory conditions; however, they remain ill-characterized. In this study, CXCL8, an inflammatory chemokine, was shown to induce the formation of filopodia-like protrusions on endothelial cells; the same effects were observed with CXCL10 and CCL5. Chemokines stimulated filopodia formation by both microvascular (from bone marrow and skin) and macrovascular (from human umbilical vein) endothelial cells. Use of blocking Abs and degradative enzymes demonstrated that CXCL8-stimulated filopodia formation was mediated by CXCR1 and CXCR2, Duffy Ag/receptor for chemokines, heparan sulfate (HS), and syndecans. HS was present on filopodial protrusions appearing as a meshwork on the cell surface, which colocalized with CXCL8, and this glycosaminoglycan was 2,6-O- and 3-O-sulfated. Transmission electron microscopy revealed that CXCL8-stimulated filopodial and microvilli-like protrusions that interacted with leukocytes before transendothelial migration and removal of HS reduced this migration. iTRAQ mass spectrometry showed that changes in the levels of cytoskeletal, signaling, and extracellular matrix proteins were associated with CXCL8-stimulated filopodia/microvilli formation; these included tropomyosin, fascin, and Rab7. This study suggests that chemokines stimulate endothelial filopodia and microvilli formation, leading to their presentation to leukocytes and leukocyte transendothelial migration.