ARAP3 protects from excessive formylated peptide-induced microvascular leakage by acting on endothelial cells and neutrophils.

Vascular permeability is temporarily heightened during inflammation, but excessive inflammation-associated microvascular leakage can be detrimental, as evidenced in the inflamed lung. Formylated peptides regulate vascular leakage indirectly via formylated peptide receptor-1 (FPR1)-mediated recruitment and activation of neutrophils. Here we identify how the GTPase-activating protein ARAP3 protects against formylated peptide-induced microvascular permeability via endothelial cells and neutrophils. In vitro, Arap3-/- endothelial monolayers were characterised by enhanced formylated peptide-induced permeability due to upregulated endothelial FPR1 and enhanced vascular endothelial cadherin internalisation. In vivo, enhanced inflammation-associated microvascular leakage was observed in Arap3-/- mice. Leakage of plasma protein into the lungs of Arap3-/- mice increased within hours of formylated peptide administration. Adoptive transfer experiments indicated this was dependent upon ARAP3 deficiency in both immune and non-immune cells. Bronchoalveolar lavages of formylated peptide-challenged Arap3-/- mice contained neutrophil extracellular traps (NETs). Pharmacological inhibition of NET formation abrogated excessive microvascular leakage, indicating a critical function of NETs in this context. The observation that Arap3-/- mice developed more severe influenza suggests these findings are pertinent to pathological situations characterised by abundant formylated peptides. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Cyclic-AMP Increases Nuclear Actin Monomer Which Promotes Proteasomal Degradation of RelA/p65 Leading to Anti-Inflammatory Effects.

The second messenger, cAMP has potent immunosuppressive and anti-inflammatory actions. These have been attributed, in part, to the ability of cAMP-induced signals to interfere with the function of the proinflammatory transcription factor Nuclear Factor-kappa B (NF-κB). However, the mechanisms underlying the modulation of NF-κB activity by cAMP remain unclear. Here we demonstrate an important role for cAMP-mediated increase in nuclear actin monomer levels in inhibiting NF-κB activity. Elevated cAMP or forced expression of a nuclear localised polymerisation defective actin mutant (NLS-ActinR62D) inhibited basal and TNFα induced mRNA levels of NF-κB-dependent genes and NF-κB-dependent reporter gene activity. Elevated cAMP or NLS-ActinR62D did not affect NF-κB nuclear translocation but did reduce total cellular and nuclear RelA/p65 levels. Preventing the cAMP-induced increase in nuclear actin monomer, either by expressing a nuclear localised active mutant of the actin polymerising protein mDIA, silencing components of the nuclear actin import complex IPO9 and CFL1 or overexpressing the nuclear export complex XPO6, rescued RelA/p65 levels and NF-κB reporter gene activity in forskolin-stimulated cells. Elevated cAMP or NLS-ActinR62D reduced the half-life of RelA/p65, which was reversed by the proteasome inhibitor MG132. Accordingly, forskolin stimulated association of RelA/p65 with ubiquitin affinity beads, indicating increased ubiquitination of RelA/p65 or associated proteins. Taken together, our data demonstrate a novel mechanism underlying the anti-inflammatory effects of cAMP and highlight the important role played by nuclear actin in the regulation of inflammation.

srGAP2 deactivates RhoA to control the duration of thrombin-mediated endothelial permeability.

The endothelial barrier is a tightly regulated gateway in the transport of material between circulation and the tissues. Inflammatory mediators such as thrombin are able to open paracellular spaces in the endothelial monolayer to allow the extravasation of plasma proteins and leukocytes. Here we show that the protein SLIT-ROBO Rho GTPase-activating protein 2 (srGAP2) plays a critical role in regulating the extent of thrombin-mediated opening. We show that srGAP2 is not required for normal barrier function in resting endothelial cells, but that depletion of srGAP2 significantly increases the magnitude and duration of junctional opening in response to thrombin. We show that srGAP2 acts to switch off RhoA signaling after the contraction phase of thrombin-induced permeability, allowing respreading of cells and reformation of the barrier. srGAP2 is also required for effective restoration of the barrier after treatment with two other vasoactive agents that active RhoA - TNFα and angiotensin II. Taken together, we show that srGAP2 has a general function in controlling RhoA signaling in endothelial permeability, acting to limit the degree and duration of opening, by triggering the switch from endothelial cell contraction to respreading.

Reduced Glomerular Filtration in Diabetes Is Attributable to Loss of Density and Increased Resistance of Glomerular Endothelial Cell Fenestrations.

BACKGROUND: Glomerular endothelial cell (GEnC) fenestrations are recognized as an essential component of the glomerular filtration barrier, yet little is known about how they are regulated and their role in disease. METHODS: We comprehensively characterized GEnC fenestral and functional renal filtration changes including measurement of glomerular Kf and GFR in diabetic mice (BTBR ob-/ob- ). We also examined and compared human samples. We evaluated Eps homology domain protein-3 (EHD3) and its association with GEnC fenestrations in diabetes in disease samples and further explored its role as a potential regulator of fenestrations in an in vitro model of fenestration formation using b.End5 cells. RESULTS: Loss of GEnC fenestration density was associated with decreased filtration function in diabetic nephropathy. We identified increased diaphragmed fenestrations in diabetes, which are posited to increase resistance to filtration and further contribute to decreased GFR. We identified decreased glomerular EHD3 expression in diabetes, which was significantly correlated with decreased fenestration density. Reduced fenestrations in EHD3 knockdown b.End5 cells in vitro further suggested a mechanistic role for EHD3 in fenestration formation. CONCLUSIONS: This study demonstrates the critical role of GEnC fenestrations in renal filtration function and suggests EHD3 may be a key regulator, loss of which may contribute to declining glomerular filtration function through aberrant GEnC fenestration regulation. This points to EHD3 as a novel therapeutic target to restore filtration function in disease.

The cancer angiogenesis co-culture assay: In vitro quantification of the angiogenic potential of tumoroids.

The treatment response to anti-angiogenic agents varies among cancer patients and predictive biomarkers are needed to identify patients with resistant cancer or guide the choice of anti-angiogenic treatment. We present "the Cancer Angiogenesis Co-Culture (CACC) assay", an in vitro Functional Precision Medicine assay which enables the study of tumouroid induced angiogenesis. This assay can quantify the ability of a patient-derived tumouroid to induce vascularization by measuring the induction of tube formation in a co-culture of vascular cells and tumoroids established from the primary colorectal tumour or a metastasis. Furthermore, the assay can quantify the sensitivity of patient-derived tumoroids to anti-angiogenic therapies. We observed that tube formation increased in a dose-dependent manner upon treatment with the pro-angiogenic factor vascular endothelial growth factor A (VEGF-A). When investigating the angiogenic potential of tumoroids from 12 patients we found that 9 tumoroid cultures induced a significant increase in tube formation compared to controls without tumoroids. In these 9 angiogenic tumoroid cultures the tube formation could be abolished by treatment with one or more of the investigated anti-angiogenic agents. The 3 non-angiogenic tumoroid cultures secreted VEGF-A but we observed no correlation between the amount of tube formation and tumoroid-secreted VEGF-A. Our data suggests that the CACC assay recapitulates the complexity of tumour angiogenesis, and when clinically verified, could prove a valuable tool to quantify sensitivity towards different anti-angiogenic agents.

A functional antagonism between RhoJ and Cdc42 regulates fibronectin remodelling during angiogenesis.

Angiogenesis is the formation of new blood vessels from pre-existing ones. Angiogenesis requires endothelial cells to change shape and polarity, as well as acquire the ability to directionally migrate ‒ processes that are classically regulated by the Rho family of GTPases. RhoJ (previously TCL) is an endothelium enriched Rho GTPase with a 78% amino acid similarity to the ubiquitously expressed Cdc42. In our recent publication, we demonstrate that α5ß1 integrin co-traffics with RhoJ. RhoJ specifically represses the internalization of the active α5ß1 conformer, leading to a reduced ability of endothelial cells to form fibronectin fibrils. Surprisingly, this function of RhoJ is in opposition to the role of Cdc42, a known driver of fibrillogenesis. Intriguingly, we discovered that the competition for limiting amounts of the shared effector, PAK3, could explain the ability of these two Rho GTPases to regulate fibrillogenesis in opposing directions. Consequently, RhoJ null mice show excessive fibronectin deposition around retinal vessels, possibly due to the unopposed action of Cdc42. Our work suggests that the functional antagonism between RhoJ and Cdc42 could restrict fibronectin remodelling to sites of active angiogenesis to form a provisional matrix for vessel growth. One correlate of our findings is that RhoJ dependent repression of fibronectin remodelling could be atheroprotective in quiescent vessels.

In Vitro Coculture Assays of Angiogenesis.

During angiogenesis, endothelial cells must undergo a coordinated set of morphological changes in order to form a new vessel. There is a need for endothelial cells to communicate with each other in order to take up different identities in the sprout and to migrate collectively as a connected chord. Endothelial cells must also interact with a wide range of other cells that contribute to vessel formation. In ischemic disease, hypoxic cells in tissue will generate proangiogenic signals that promote and guide angiogenesis. In solid tumors, this function is co-opted by tumor cells, which make a complex range of interactions with endothelial cells, even integrating into the walls of vessels. In vessel repair, cells from the immune system contribute to the promotion and remodeling of new vessels. The coculture angiogenesis assay is a long-term in vitro protocol that uses fibroblasts to secrete and condition an artificial stromal matrix for tubules to grow through. We show here how the assay can be easily adapted to include additional cell types, facilitating the study of cellular interactions during neovascularization.

Dimethyl-2-oxoglutarate improves redox balance and mitochondrial function in muscle pericytes of individuals with diabetes mellitus.

AIMS/HYPOTHESIS: Treatment of vascular complications of diabetes remains inadequate. We reported that muscle pericytes (MPs) from limb muscles of vascular patients with diabetes mellitus display elevated levels of oxidative stress causing a dysfunctional phenotype. Here, we investigated whether treatment with dimethyl-2-oxoglutarate (DM-2OG), a tricarboxylic acid cycle metabolite with antioxidant properties, can restore a healthy metabolic and functional phenotype. METHODS: MPs were isolated from limb muscles of diabetes patients with vascular disease (D-MPs) and from non-diabetic control participants (ND-MPs). Metabolic status was assessed in untreated and DM-2OG-treated (1 mmol/l) cells using an extracellular flux analyser and anion-exchange chromatography-mass spectrometry (IC-MS/MS). Redox status was measured using commercial kits and IC-MS/MS, with antioxidant and metabolic enzyme expression assessed by quantitative RT-PCR and western blotting. Myogenic differentiation and proliferation and pericyte-endothelial interaction were assessed as functional readouts. RESULTS: D-MPs showed mitochondrial dysfunction, suppressed glycolytic activity and reduced reactive oxygen species-buffering capacity, but no suppression of antioxidant systems when compared with ND-MP controls. DM-2OG supplementation improved redox balance and mitochondrial function, without affecting glycolysis or antioxidant systems. Nonetheless, this was not enough for treated D-MPs to regain the level of proliferation and myogenic differentiation of ND-MPs. Interestingly, DM-2OG exerted a positive effect on pericyte-endothelial cell interaction in the co-culture angiogenesis assay, independent of the diabetic status. CONCLUSIONS/INTERPRETATION: These novel findings support the concept of using DM-2OG supplementation to improve pericyte redox balance and mitochondrial function, while concurrently allowing for enhanced pericyte-endothelial crosstalk. Such effects may help to prevent or slow down vasculopathy in skeletal muscles of people with diabetes. Graphical abstract.

RhoJ Regulates α5ß1 Integrin Trafficking to Control Fibronectin Remodeling during Angiogenesis.

Rho guanosine triphosphatases (GTPases) are master regulators of cell shape and cell movement [1]. The archetypal family members RhoA, Rac1, and Cdc42 arose early in eukaryotic evolution and coordinate a diverse range of cell morphologies and migrations. Evolution of the vertebrates was paralleled by expansion of this family through gene duplication. Emergence of an adaptive immune system and more complex neural systems presented new roles for Rho GTPases, filled by new family members. Cdc42 underwent gene duplication to produce two related proteins-RhoQ and RhoJ [2]. RhoQ is active in neural dynamics; however, RhoJ is highly expressed in endothelial cells under control of the endothelial-specific promoter ERG [3, 4]. RhoJ is required for angiogenesis [5, 6] and has multiple roles in this process [7, 8]. We recently demonstrated that RhoJ regulates the endosomal trafficking of podocalyxin during angiogenesis to control lumen formation [9]. Here, we use vesicle purification and proteomic analysis to identify the endothelial targets of RhoJ-mediated trafficking. We identify α5ß1 integrin as a major RhoJ cargo and show that RhoJ regulates the intracellular trafficking of active α5ß1 integrin in endothelial cells to repress fibronectin fibrillogenesis. Accordingly, mice lacking RhoJ show deregulated deposition of fibronectin around vessels during developmental angiogenesis. Intriguingly, we show that RhoJ acts in opposition to Cdc42 in this process through competition for a shared partner, PAK3. These studies identify a critical role for RhoJ in matrix remodeling during blood vessel formation and demonstrate a functional interrelationship between RhoJ and its evolutionary parent.

Raftlin is recruited by neuropilin-1 to the activated VEGFR2 complex to control proangiogenic signaling.

BACKGROUND: VEGFR2 (vascular endothelial growth factor receptor 2) is the major pro-angiogenic receptor in endothelial cells. Compared to other members of the receptor tyrosine kinase family, we know relatively few VEGFR2 signaling partners. Our objective was to use mass spectrometry-based proteomics to identify novel binding partners of activated VEGFR2. METHODS: We created an endothelial cell line stably expressing GFP-tagged VEGFR2 and isolated activated receptor complexes. Analysis by mass spectrometry identified raftlin as a novel binding partner of VEGFR2. RESULTS: We found that raftlin is recruited to the activated VEGFR2 complex via the co-receptor Nrp1 (neuropilin-1). We show that raftlin regulates the surface levels of Nrp1 in endothelial cells, controlling the availability of Nrp1 for VEGFR2 interaction. Raftlin stabilizes active VEGFR2 at the cell surface by inhibiting endocytosis of the activated receptor. Raftlin also promotes recycling of internalized VEGFR2 to the cell surface. Raftlin alters the signaling outcomes of VEGFR2 activation, inhibiting the activation of p38 and FAK (focal adhesion kinases) specifically. Both pathways are linked to cell migration in endothelial cells, and raftlin inhibits endothelial cell migration in response to VEGF. CONCLUSION: Nrp1 is an important co-receptor for VEGFR2; however, its functions are still only partially understood. We show that raftlin works with Nrp1 in endothelial cells to control intracellular trafficking of the activated VEGFR2. This modulates the response to VEGF and controls endothelial cell migration.

Characterization of the polarized endothelial secretome.

Endothelial cells (ECs) form an active barrier between the circulation and the body. In addition to controlling transport of molecules between these 2 compartments, the endothelium is a major secretory organ, releasing proteins both into the circulation and into the vascular matrix. Although it is clearly important that proteins are correctly sorted into these 2 spaces, we currently know little of the polarization of this secretion or how it is controlled. Here, we present an optimized system for the analysis of polarized secretion and show that it allows the derivation of deep, robust proteomes from small numbers of primary ECs. We present the first endothelial apically and basolaterally secreted proteomes, demonstrating that ECs polarize the secretion of extracellular vesicle cargoes to the apical surface. Conversely, we find that protein secretion at the basolateral surface is focused on components of the extracellular matrix (ECM). Finally, we examine the role of liprin-α1 in secretion toward the basolateral compartment and identify a subset of ECM components that share this route with fibronectin.-Wei, H., Sundararaman, A., Dickson, E., Rennie-Campbell, L., Cross, E., Heesom, K. J., Mellor, H. Characterization of the polarized endothelial secretome.

Live imaging of wound angiogenesis reveals macrophage orchestrated vessel sprouting and regression.

Wound angiogenesis is an integral part of tissue repair and is impaired in many pathologies of healing. Here, we investigate the cellular interactions between innate immune cells and endothelial cells at wounds that drive neoangiogenic sprouting in real time and in vivo Our studies in mouse and zebrafish wounds indicate that macrophages are drawn to wound blood vessels soon after injury and are intimately associated throughout the repair process and that macrophage ablation results in impaired neoangiogenesis. Macrophages also positively influence wound angiogenesis by driving resolution of anti-angiogenic wound neutrophils. Experimental manipulation of the wound environment to specifically alter macrophage activation state dramatically influences subsequent blood vessel sprouting, with premature dampening of tumour necrosis factor-α expression leading to impaired neoangiogenesis. Complementary human tissue culture studies indicate that inflammatory macrophages associate with endothelial cells and are sufficient to drive vessel sprouting via vascular endothelial growth factor signalling. Subsequently, macrophages also play a role in blood vessel regression during the resolution phase of wound repair, and their absence, or shifted activation state, impairs appropriate vessel clearance.

Direct Activation of NADPH Oxidase 2 by 2-Deoxyribose-1-Phosphate Triggers Nuclear Factor Kappa B-Dependent Angiogenesis.

AIMS: Deoxyribose-1-phosphate (dRP) is a proangiogenic paracrine stimulus released by cancer cells, platelets, and macrophages and acting on endothelial cells. The objective of this study was to clarify how dRP stimulates angiogenic responses in human endothelial cells. RESULTS: Live cell imaging, electron paramagnetic resonance, pull-down of dRP-interacting proteins, followed by immunoblotting, gene silencing of different NADPH oxidases (NOXs), and their regulatory cosubunits by small interfering RNA (siRNA) transfection, and experiments with inhibitors of the sugar transporter glucose transporter 1 (GLUT1) were utilized to demonstrate that dRP acts intracellularly by directly activating the endothelial NOX2 complex, but not NOX4. Increased reactive oxygen species generation in response to NOX2 activity leads to redox-dependent activation of the transcription factor nuclear factor kappa B (NF-κB), which, in turn, induces vascular endothelial growth factor receptor 2 (VEGFR2) upregulation. Using endothelial tube formation assays, gene silencing by siRNA, and antibody-based receptor inhibition, we demonstrate that the activation of NF-κB and VEGFR2 is necessary for the angiogenic responses elicited by dRP. The upregulation of VEGFR2 and NOX2-dependent stimulation of angiogenesis by dRP were confirmed in excisional wound and Matrigel plug vascularization assays in vivo using NOX2-/- mice. INNOVATION: For the first time, we demonstrate that dRP acts intracellularly and stimulates superoxide anion generation by direct binding and activation of the NOX2 enzymatic complex. CONCLUSIONS: This study describes a novel molecular mechanism underlying the proangiogenic activity of dRP, which involves the sequential activation of NOX2 and NF-κB and upregulation of VEGFR2. Antioxid. Redox Signal. 28, 110-130.

The tumor suppressor RhoBTB1 controls Golgi integrity and breast cancer cell invasion through METTL7B.

BACKGROUND: RhoBTB1 and 2 are atypical members of the Rho GTPase family of signaling proteins. Unlike other Rho GTPases, RhoBTB1 and 2 undergo silencing or mutation in a wide range of epithelial cancers; however, little is known about the consequences of this loss of function. METHODS: We analyzed transcriptome data to identify transcriptional targets of RhoBTB2. We verified these using Q-PCR and then used gene silencing and cell imaging to determine the cellular function of these targets downstream of RhoBTB signaling. RESULTS: RhoBTB1 and 2 regulate the expression of the methyltransferases METTL7B and METTL7A, respectively. RhoBTB1 regulates the integrity of the Golgi complex through METTL7B. RhoBTB1 is required for expression of METTL7B and silencing of either protein leads to fragmentation of the Golgi. Loss of RhoBTB1 expression is linked to Golgi fragmentation in breast cancer cells. Restoration of normal RhoBTB1 expression rescues Golgi morphology and dramatically inhibits breast cancer cell invasion. CONCLUSION: Loss of RhoBTB1 expression in breast cancer cells leads to Golgi fragmentation and hence loss of normal polarity.

In Vitro Coculture Assays of Angiogenesis.

During angiogenesis, endothelial cells invade into the stromal matrix: a complex, structured array of extracellular matrix proteins. This three-dimensional deformable substrate also contains a mixture of angiogenic factors as well as embedded stromal cells. Interactions between endothelial cells and the stromal tissue make complex and important contributions to the process of angiogenesis; however, the composition of the stromal matrix is hard to replicate in vitro. The coculture angiogenesis assay is a long-term assay that uses fibroblasts to secrete and condition a stromal matrix that more closely mimics tissue than a simple collagen gel. Like all in vitro assays of angiogenesis, it has both strengths and weaknesses. Here we give protocols for the two of the most useful applications of the assay: screening for regulators of angiogenesis and high-resolution imaging.

The Formin FMNL3 Controls Early Apical Specification in Endothelial Cells by Regulating the Polarized Trafficking of Podocalyxin.

Angiogenesis is the fundamental process by which new blood vessels form from pre-existing vasculature. It plays a critical role in the formation of the vasculature during development and is triggered in response to tissue hypoxia in adult organisms. This process requires complex and coordinated regulation of the endothelial cell cytoskeleton to control cell shape and polarity. In our previous work, we showed that the cytoskeletal regulator FMNL3/FRL2 controls the alignment of stabilized microtubules during polarized endothelial cell elongation and that depletion of FMNL3 retards elongation of the intersegmental vessels in zebrafish. Recent work has shown that FMNL3 is also needed for vascular lumen formation, a critical element of the formation of functional vessels. Here, we show that FMNL3 interacts with Cdc42 and RhoJ, two Rho family GTPases known to be required for lumen formation. FMNL3 and RhoJ are concentrated at the early apical surface, or AMIS, and regulate the formation of radiating actin cables from this site. In diverse biological systems, formins mediate polarized trafficking through the generation of similar actin filaments tracks. We show that FMNL3 and RhoJ are required for polarized trafficking of podocalyxin to the early apical surface--an important event in vascular lumenogenesis.

Platelet Rho GTPases-a focus on novel players, roles and relationships.

Rho GTPases are critical for platelet function. Although the roles of RhoA, Rac and Cdc42 are characterized, platelets express other Rho GTPases, whose activities are less well understood. This review summarizes our understanding of the roles of platelet Rho GTPases and focuses particularly on the functions of Rif and RhoG. In human platelets, Rif interacts with cytoskeleton regulators including formins mDia1 and mDia3, whereas RhoG binds SNARE-complex proteins and cytoskeletal regulators ELMO and DOCK1. Knockout mouse studies suggest that Rif plays no critical functions in platelets, likely due to functional overlap with other Rho GTPases. In contrast, RhoG is essential for normal granule secretion downstream of the collagen receptor GPVI. The central defect in RhoG-/- platelets is reduced dense granule secretion, which impedes integrin activation and aggregation and limits platelet recruitment to growing thrombi under shear, translating into reduced thrombus formation in vivo. Potential avenues for future work on Rho GTPases in platelets are also highlighted, including identification of the key regulator for platelet filopodia formation and investigation of the role of the many Rho GTPase regulators in platelet function in both health and disease.

The Rif GTPase regulates cytoskeletal signaling from plexinA4 to promote neurite retraction.

The small GTPase Rif is required for the early stages of dendritic spine formation in neurons, acting through the formin mDia2 to control actin polymerization. Rif is expressed at high levels in the brain, suggesting broader roles in neuronal function. We screened a yeast two-hybrid cDNA library to identify additional binding partners for Rif of potential relevance to neuronal function. We found that Rif interacts with FARP1, a neuronal activator of the RhoA GTPase. We show that Rif has two separate roles in FARP1 regulation-in controlling its association with plexinA4, and in releasing active RhoA from a plexinA4/FARP1 complex. The regulation of FARP1 by Rif promotes neurite retraction in cells stimulated with the semaphorin Sema6A.

The coculture organotypic assay of angiogenesis.

Angiogenesis is a complex process involving the interactions of endothelial cells not only with pro-angiogenic factors but also with stromal cells and stromal matrix components. Modeling this process in vitro is challenging, and many different assays have been described, each with their own particular strengths and weaknesses. The coculture assay is a long-term assay of angiogenesis that uses fibroblasts to secrete and condition a stromal matrix that more closely mimics tissue than a simple collagen gel. The assay is particularly suited to screening for angiogenic regulators and also for high-resolution imaging of endothelial cells undergoing angiogenic morphogenesis.

RNA interference approaches to examine Golgi function in animal cell culture.

The ability to deplete specific proteins from cells has transformed cell biology. Targeting of gene transcripts using RNA interference has allowed for a highly refined approach to the analysis of gene function that has been applied to all aspects of cell biology. Developments of the technology have reached a point where it is now a relatively trivial task to assess the role of an individual protein in a particular cell function. RNAi also allows for genome-wide screening as a discovery step toward the identification of new components of cellular pathways and machines. The technique has been applied extensively to the analysis of Golgi complex function, leading to significant insight into the biology of this complex organelle. Here, we describe the commonly used options for targeting individual genes for both transient and stable knockdown. We consider the alternative methods for introducing these reagents into cells and outline methods that we and others have used widely for validation of specificity and efficacy of gene targeting.