Neurotrophic extracellular matrix proteins promote neuronal and iPSC astrocyte progenitor cell- and nano-scale process extension for neural repair applications.

The extracellular matrix plays a critical role in modulating cell behaviour in the developing and adult central nervous system influencing neural cell morphology, function and growth. Neurons and astrocytes, play vital roles in neural signalling and support respectively and respond to cues from the surrounding matrix environment. However, a better understanding of the impact of specific individual extracellular matrix proteins on both neurons and astrocytes is critical for advancing the development of matrix-based scaffolds for neural repair applications. This study aimed to provide an in-depth analysis of how different commonly used extracellular matrix proteins- laminin-1, Fn, collagen IV, and collagen I-affect the morphology and growth of trophic induced pluripotent stem cell (iPSC)-derived astrocyte progenitors and mouse motor neuron-like cells. Following a 7-day culture period, morphological assessments revealed that laminin-1, fibronectin, and collagen-IV, but not collagen I, promoted increased process extension and a stellate morphology in astrocytes, with collagen-IV yielding the greatest increases. Subsequent analysis of neurons grown on the different extracellular matrix proteins revealed a similar pattern with laminin-1, fibronectin, and collagen-IV supporting robust neurite outgrowth. fibronectin promoted the greatest increase in neurite extension, while collagen-I did not enhance neurite growth compared to poly-L-lysine controls. Super-resolution microscopy highlighted extracellular matrix-specific nanoscale changes in cytoskeletal organization, with distinct patterns of actin filament distribution where the three basement membrane-associated proteins (laminin-1, fibronectin, and collagen-IV) promoted the extension of fine cellular processes. Overall, this study demonstrates the potent effect of laminin-1, fibronectin and collagen-IV to promote both iPSC-derived astrocyte progenitor and neuronal growth, yielding detailed insights into the effect of extracellular matrix proteins on neural cell morphology at both the whole cell and nanoscale levels. The ability of laminin-1, collagen-IV and fibronectin to elicit strong growth-promoting effects highlight their suitability as optimal extracellular matrix proteins to incorporate into neurotrophic biomaterial scaffolds for the delivery of cell cargoes for neural repair.

VEGFD/VEGFR3 signaling contributes to the dysfunction of the astrocyte IL-3/microglia IL-3Rα cross-talk and drives neuroinflammation in mouse ischemic stroke.

Astrocyte-derived IL-3 activates the corresponding receptor IL-3Rα in microglia. This cross-talk between astrocytes and microglia ameliorates the pathology of Alzheimer's disease in mice. In this study we investigated the role of IL-3/IL-3Rα cross-talk and its regulatory mechanisms in ischemic stroke. Ischemic stroke was induced in mice by intraluminal occlusion of the right middle cerebral artery (MCA) for 60 min followed by reperfusion (I/R). Human astrocytes or microglia subjected to oxygen-glucose deprivation and reoxygenation (OGD/Re) were used as in vitro models of brain ischemia. We showed that both I/R and OGD/Re significantly induced decreases in astrocytic IL-3 and microglial IL-3Rα protein levels, accompanied by pro-inflammatory activation of A1-type astrocytes and M1-type microglia. Importantly, astrocyte-derived VEGFD acting on VEGFR3 of astrocytes and microglia contributed to the cross-talk dysfunction and pro-inflammatory activation of the two glial cells, thereby mediating neuronal cell damage. By using metabolomics and multiple biochemical approaches, we demonstrated that IL-3 supplementation to microglia reversed OGD/Re-induced lipid metabolic reprogramming evidenced by upregulated expression of CPT1A, a rate-limiting enzyme for the mitochondrial ß-oxidation, and increased levels of glycerophospholipids, the major components of cellular membranes, causing reduced accumulation of lipid droplets, thus reduced pro-inflammatory activation and necrosis, as well as increased phagocytosis of microglia. Notably, exogenous IL-3 and the VEGFR antagonist axitinib reestablished the cross-talk of IL-3/IL-3Rα, improving microglial lipid metabolic levels via upregulation of CPT1A, restoring microglial phagocytotic function and attenuating microglial pro-inflammatory activation, ultimately contributing to brain recovery from I/R insult. Our results demonstrate that VEGFD/VEGFR3 signaling contributes to the dysfunction of the astrocyte IL-3/microglia IL-3Rα cross-talk and drives pro-inflammatory activation, causing lipid metabolic reprogramming of microglia. These insights suggest VEGFR3 antagonism or restoring IL-3 levels as a potential therapeutic strategy for ischemic stroke.

O-glycan determinants regulate VWF trafficking to Weibel-Palade bodies.

ABSTRACT: von Willebrand factor (VWF) undergoes complex posttranslational modification within endothelial cells (ECs) before secretion. This includes significant N- and O-linked glycosylation. Previous studies have demonstrated that changes in N-linked glycan structures significantly influence VWF biosynthesis. In contrast, although abnormalities in VWF O-linked glycans (OLGs) have been associated with enhanced VWF clearance, their effect on VWF biosynthesis remains poorly explored. Herein, we report a novel role for OLG determinants in regulating VWF biosynthesis and trafficking within ECs. We demonstrate that alterations in OLGs (notably reduced terminal sialylation) lead to activation of the A1 domain of VWF within EC. In the presence of altered OLG, VWF multimerization is reduced and Weibel-Palade body (WPB) formation significantly impaired. Consistently, the amount of VWF secreted from WPB after EC activation was significantly reduced in the context of O-glycosylation inhibition. Finally, altered OLG on VWF not only reduced the amount of VWF secreted after EC activation but also affected its hemostatic efficacy. Notably, VWF secreted after WPB exocytosis consisted predominantly of low molecular weight multimers, and the length of tethered VWF string formation on the surface of activated ECs was significantly reduced. In conclusion, our data therefore support the hypothesis that alterations in O-glycosylation pathways directly affect VWF trafficking within human EC. These findings are interesting given that previous studies have reported altered OLG on plasma VWF (notably increased T-antigen expression) in patients with von Willebrand disease.

Corrigendum to Contractility defects hinder glycoprotein VI-mediated platelet activation and affect platelet functions beyond clot contraction [Research and Practice in Thrombosis and Haemostasis Volume 8, Issue 1, January 2024, 102322].

[This corrects the article DOI: 10.1016/j.rpth.2024.102322.].

A comprehensive program in academic health professions to enhance faculty recruitment, mentoring, productivity, and retention: The case of RCSI's StAR program.

Top faculty talent recruitment, mentoring, productivity, and retention are paramount for organizational success among institutions of higher learning. Programs would do well to treat these various aspects of faculty management/development as inextricably linked to one another, rather than viewing recruitment or retention in a vacuum. The Strategic Academic Recruitment (StAR) program at the Royal College of Surgeons in Ireland (RCSI) University of Medicine and Health Sciences in Dublin was founded to bear these things, along with best practices in faculty development, in mind to enhance organizational effectiveness. This paper provides some background, description, and outcomes of the program thus far, revealing positive trends in scholarly productivity, teaching, program faculty commitment, and the development of future leaders for the institution, even while further evaluation and continued quality improvement for the StAR initiative are called for. It is hoped that the details provided here can be helpful for other academic organizations as they consider any of various initiatives aimed to attract high-quality labor capital, position those faculty for success, and enhance organizational effectiveness and reputation.

Contractility defects hinder glycoprotein VI-mediated platelet activation and affect platelet functions beyond clot contraction.

Background: Active and passive biomechanical properties of platelets contribute substantially to thrombus formation. Actomyosin contractility drives clot contraction required for stabilizing the hemostatic plug. Impaired contractility results in bleeding but is difficult to detect using platelet function tests. Objectives: To determine how diminished myosin activity affects platelet functions, including and beyond clot contraction. Methods: Using the myosin IIA-specific pharmacologic inhibitor blebbistatin, we modulated myosin activity in platelets from healthy donors and systematically characterized platelet responses at various levels of inhibition by interrogating distinct platelet functions at each stage of thrombus formation using a range of complementary assays. Results: Partial myosin IIA inhibition neither affected platelet von Willebrand factor interactions under arterial shear nor platelet spreading and cytoskeletal rearrangements on fibrinogen. However, it impacted stress fiber formation and the nanoarchitecture of cell-matrix adhesions, drastically reducing and limiting traction forces. Higher blebbistatin concentrations impaired platelet adhesion under flow, altered mechanosensing at lamellipodia edges, and eliminated traction forces without affecting platelet spreading, α-granule secretion, or procoagulant platelet formation. Unexpectedly, myosin IIA inhibition reduced calcium influx, dense granule secretion, and platelet aggregation downstream of glycoprotein (GP)VI and limited the redistribution of GPVI on the cell membrane, whereas aggregation induced by adenosine diphosphate or arachidonic acid was unaffected. Conclusion: Our findings highlight the importance of both active contractile and passive crosslinking roles of myosin IIA in the platelet cytoskeleton. They support the hypothesis that highly contractile platelets are needed for hemostasis and further suggest a supportive role for myosin IIA in GPVI signaling.

Longevity interventions modulate mechanotransduction and extracellular matrix homeostasis in C. elegans.

Dysfunctional extracellular matrices (ECM) contribute to aging and disease. Repairing dysfunctional ECM could potentially prevent age-related pathologies. Interventions promoting longevity also impact ECM gene expression. However, the role of ECM composition changes in healthy aging remains unclear. Here we perform proteomics and in-vivo monitoring to systematically investigate ECM composition (matreotype) during aging in C. elegans revealing three distinct collagen dynamics. Longevity interventions slow age-related collagen stiffening and prolong the expression of collagens that are turned over. These prolonged collagen dynamics are mediated by a mechanical feedback loop of hemidesmosome-containing structures that span from the exoskeletal ECM through the hypodermis, basement membrane ECM, to the muscles, coupling mechanical forces to adjust ECM gene expression and longevity via the transcriptional co-activator YAP-1 across tissues. Our results provide in-vivo evidence that coordinated ECM remodeling through mechanotransduction is required and sufficient to promote longevity, offering potential avenues for interventions targeting ECM dynamics.

Glycolytic reprogramming fuels myeloid cell-driven hypercoagulability.

BACKGROUND: Myeloid cell metabolic reprogramming is a hallmark of inflammatory disease; however, its role in inflammation-induced hypercoagulability is poorly understood. OBJECTIVES: We aimed to evaluate the role of inflammation-associated metabolic reprogramming in regulating blood coagulation. METHODS: We used novel myeloid cell-based global hemostasis assays and murine models of immunometabolic disease. RESULTS: Glycolysis was essential for enhanced activated myeloid cell tissue factor expression and decryption, driving increased cell-dependent thrombin generation in response to inflammatory challenge. Similarly, inhibition of glycolysis enhanced activated macrophage fibrinolytic activity through reduced plasminogen activator inhibitor 1 activity. Macrophage polarization or activation markedly increased endothelial protein C receptor (EPCR) expression on monocytes and macrophages, leading to increased myeloid cell-dependent protein C activation. Importantly, inflammation-dependent EPCR expression on tissue-resident macrophages was also observed in vivo. Adipose tissue macrophages from obese mice fed a high-fat diet exhibited significantly enhanced EPCR expression and activated protein C generation compared with macrophages isolated from the adipose tissue of healthy mice. Similarly, the induction of colitis in mice prompted infiltration of EPCR+ innate myeloid cells within inflamed colonic tissue that were absent from the intestinal tissue of healthy mice. CONCLUSION: Collectively, this study identifies immunometabolic regulation of myeloid cell hypercoagulability, opening new therapeutic possibilities for targeted mitigation of thromboinflammatory disease.

Platelet mechanosensing as key to understanding platelet function.

PURPOSE OF REVIEW: This review highlights how the perception of platelet function is evolving based on recent insights into platelet mechanobiology. RECENT FINDINGS: The mechanosensitive ion channel Piezo1 mediates activation of free-flowing platelets under conditions of flow acceleration through mechanisms independent of adhesion receptors and classical activation pathways. Interference with the initiation of platelet migration or with the phenotypic switch of migrating platelets to a procoagulant state aggravates inflammatory bleeding. Mechanosensing of biochemical and biophysical microenvironmental cues during thrombus formation feed into platelet contractile force generation. Measurements of single platelet contraction and bulk clot retraction show promise to identify individuals at risk for hemorrhage. SUMMARY: New findings unravel novel mechanotransduction pathways and effector functions in platelets, establishing mechanobiology as a pivotal component of platelet function. These insights highlight limitations of existing treatments and offer new potential therapeutic approaches and diagnostic avenues based on mechanobiological principles. Further extensive research is required to distinguish between core hemostatic and pathological mechanisms influenced by platelet mechanosensing.

RhoGAP6 interacts with COPI to regulate protein transport.

RhoGAP6 is the most highly expressed GTPase-activating protein (GAP) in platelets specific for RhoA. Structurally RhoGAP6 contains a central catalytic GAP domain surrounded by large, disordered N- and C-termini of unknown function. Sequence analysis revealed three conserved consecutive overlapping di-tryptophan motifs close to the RhoGAP6 C-terminus which were predicted to bind to the mu homology domain (MHD) of δ-COP, a component of the COPI vesicle complex. We confirmed an endogenous interaction between RhoGAP6 and δ-COP in human platelets using GST-CD2AP which binds an N-terminal RhoGAP6 SH3 binding motif. Next, we confirmed that the MHD of δ-COP and the di-tryptophan motifs of RhoGAP6 mediate the interaction between both proteins. Each of the three di-tryptophan motifs appeared necessary for stable δ-COP binding. Proteomic analysis of other potential RhoGAP6 di-tryptophan motif binding partners indicated that the RhoGAP6/δ-COP interaction connects RhoGAP6 to the whole COPI complex. 14-3-3 was also established as a RhoGAP6 binding partner and its binding site was mapped to serine 37. We provide evidence of potential cross-regulation between 14-3-3 and δ-COP binding, however, neither δ-COP nor 14-3-3 binding to RhoGAP6 impacted RhoA activity. Instead, analysis of protein transport through the secretory pathway demonstrated that RhoGAP6/δ-COP binding increased protein transport to the plasma membrane, as did a catalytically inactive mutant of RhoGAP6. Overall, we have identified a novel interaction between RhoGAP6 and δ-COP which is mediated by conserved C-terminal di-tryptophan motifs, and which might control protein transport in platelets.

Nanotopography of Polystyrene/Poly(methyl methacrylate) for the Promotion of Patient Specific Von Willebrand Factor Entrapment and Platelet Adhesion in a Whole Blood Microfluidic Assay.

Platelet function testing is essential for the diagnosis of patients with bleeding disorders. Specifically, there is a need for a whole blood assay that is capable of analysing platelet behaviour in contact with a patient-specific autologous von Willebrand factor (vWF), under physiologically relevant conditions. The creation of surface topography capable of entrapping and uncoiling vWF for the support of subsequent platelet adhesion within the same blood sample offers a potential basis for such an assay. In this study, spin coating of polystyrene/poly (methyl methacrylate) (PS/PMMA) demixed solutions onto glass substrates in air has been used to attain surfaces with well-defined topographical features. The effect of augmenting the PS/PMMA solution with uniform 50 µm PS microspheres that can moderate the demixing process on the resultant surface features has also been investigated. The topographical features created here by spin coating under ambient air pressure conditions, rather than in nitrogen, which previous work reports, produces substrate surfaces with the ability to entrap vWF from flowing blood and facilitate platelet adhesion. The direct optical visualisation of fluorescently-labelled platelets indicates that topography resulting from inclusion of PS microspheres in the PS/PMMA spin coating solution increases the total number of platelets that adhere to the substrate surface over the period of the microfluidic assay. However, a detailed analysis of the adhesion rate, mean translocating velocity, mean translocation distance, and fraction of the stably adhered platelets measured during blood flow under arterial equivalent mechanical shear conditions indicates no significant difference for topographies created with or without inclusion of the PS microspheres.

The circadian clock influences T cell responses to vaccination by regulating dendritic cell antigen processing.

Dendritic cells play a key role in processing and presenting antigens to naïve T cells to prime adaptive immunity. Circadian rhythms are known to regulate many aspects of immunity; however, the role of circadian rhythms in dendritic cell function is still unclear. Here, we show greater T cell responses when mice are immunised in the middle of their rest versus their active phase. We find a circadian rhythm in antigen processing that correlates with rhythms in both mitochondrial morphology and metabolism, dependent on the molecular clock gene, Bmal1. Using Mdivi-1, a compound that promotes mitochondrial fusion, we are able to rescue the circadian deficit in antigen processing and mechanistically link mitochondrial morphology and antigen processing. Furthermore, we find that circadian changes in mitochondrial Ca2+ are central to the circadian regulation of antigen processing. Our results indicate that rhythmic changes in mitochondrial calcium, which are associated with changes in mitochondrial morphology, regulate antigen processing.

von Willebrand factor links primary hemostasis to innate immunity.

The plasma multimeric glycoprotein von Willebrand factor (VWF) plays a critical role in primary hemostasis by tethering platelets to exposed collagen at sites of vascular injury. Recent studies have identified additional biological roles for VWF, and in particular suggest that VWF may play an important role in regulating inflammatory responses. However, the molecular mechanisms through which VWF exerts its immuno-modulatory effects remain poorly understood. In this study, we report that VWF binding to macrophages triggers downstream MAP kinase signaling, NF-κB activation and production of pro-inflammatory cytokines and chemokines. In addition, VWF binding also drives macrophage M1 polarization and shifts macrophage metabolism towards glycolysis in a p38-dependent manner. Cumulatively, our findings define an important biological role for VWF in modulating macrophage function, and thereby establish a novel link between primary hemostasis and innate immunity.

Breast cancer cells mediate endothelial cell activation, promoting von Willebrand factor release, tumor adhesion, and transendothelial migration.

BACKGROUND: Breast cancer results in a three- to four-fold increased risk of venous thromboembolism (VTE), which is associated with reduced patient survival. Despite this, the mechanisms underpinning breast cancer-associated thrombosis remain poorly defined. Tumor cells can trigger endothelial cell (EC) activation resulting in increased von Willebrand factor (VWF) secretion. Importantly, elevated plasma VWF levels constitute an independent biomarker for VTE risk. Moreover, in a model of melanoma, treatment with low molecular weight heparin (LMWH) negatively regulated VWF secretion and attenuated tumor metastasis. OBJECTIVE: To investigate the role of VWF in breast cancer metastasis and examine the effect of LMWH in modulating EC activation and breast tumor transmigration. METHODS: von Willebrand factor levels were measured by ELISA. Primary ECs were used to assess tumor-induced activation, angiogenesis, tumor adhesion, and transendothelial migration. RESULTS AND CONCLUSION: Patients with metastatic breast cancer have markedly elevated plasma VWF:Ag levels that also correlate with poorer survival. MDA-MB-231 and MCF-7 breast cancer cells induce secretion of VWF, angiopoietin-2, and osteoprotegerin from ECs, which is further enhanced by the presence of platelets. Vascular endothelial growth factor-A (VEGF-A) plays an important role in modulating breast cancer-induced VWF release. Moreover, VEGF-A from breast tumor cells also contributes to a pro-angiogenic effect on ECs. VWF multimers secreted from ECs, in response to tumor-VEGF-A, mediate adhesion of breast tumor cells along the endothelium. LMWH inhibits VWF-breast tumor adhesion and transendothelial migration. Our findings highlight the significant crosstalk between tumor cells and the endothelium including increased VWF secretion which may contribute to tumor metastasis.

Reduced platelet forces underlie impaired hemostasis in mouse models of MYH9-related disease.

MYH9-related disease patients with mutations in the contractile protein nonmuscle myosin heavy chain IIA display, among others, macrothrombocytopenia and a mild-to-moderate bleeding tendency. In this study, we used three mouse lines, each with one point mutation in the Myh9 gene at positions 702, 1424, or 1841, to investigate mechanisms underlying the increased bleeding risk. Agonist-induced activation of Myh9 mutant platelets was comparable to controls. However, myosin light chain phosphorylation after activation was reduced in mutant platelets, which displayed altered biophysical characteristics and generated lower adhesion, interaction, and traction forces. Treatment with tranexamic acid restored clot retraction in the presence of tPA and reduced bleeding. We verified our findings from the mutant mice with platelets from patients with the respective mutation. These data suggest that reduced platelet forces lead to an increased bleeding tendency in patients with MYH9-related disease, and treatment with tranexamic acid can improve the hemostatic function.

Nanofiber Topographies Enhance Platelet-Fibrinogen Scaffold Interactions.

The initial contact with blood and its components, including plasma proteins and platelets, directs the body's response to foreign materials. Natural scaffolds of extracellular matrix or fibrin contain fibrils with nanoscale dimensions, but how platelets specifically respond to the topography and architecture of fibrous materials is still incompletely understood. Here, planar and nanofiber scaffolds are fabricated from native fibrinogen to characterize the morphology of adherent platelets and activation markers for phosphatidylserine exposure and α-granule secretion by confocal fluorescence microscopy and scanning electron microscopy. Different fibrinogen topographies equally support the spreading and α-granule secretion of washed platelets. In contrast, preincubation of the scaffolds with plasma diminishes platelet spreading on planar fibrinogen surfaces but not on nanofibers. The data show that the enhanced interactions of platelets with nanofibers result from a higher locally accessible surface area, effectively increasing the ligand density for integrin-mediated responses. Overall, fibrinogen nanofibers direct platelets toward robust adhesion formation and α-granule secretion while minimizing their procoagulant activity. Similar results on fibrinogen-coated polydimethylsiloxane substrates with micrometer-sized 3D features suggest that surface topography could be used more generally to steer blood-materials interactions on different length scales for enhancing the initial wound healing steps.

Platelets drive fibronectin fibrillogenesis using integrin αIIbß3.

Platelets interact with multiple adhesion proteins during thrombogenesis, yet little is known about their ability to assemble fibronectin matrix. In vitro three-dimensional superresolution microscopy complemented by biophysical and biochemical methods revealed fundamental insights into how platelet contractility drives fibronectin fibrillogenesis. Platelets adhering to thrombus proteins (fibronectin and fibrin) versus basement membrane components (laminin and collagen IV) pull fibronectin fibrils along their apical membrane versus underneath their basal membrane, respectively. In contrast to other cell types, platelets assemble fibronectin nanofibrils using αIIbß3 rather than α5ß1 integrins. Apical fibrillogenesis correlated with a stronger activation of integrin-linked kinase, higher platelet traction forces, and a larger tension in fibrillar-like adhesions compared to basal fibrillogenesis. Our findings have potential implications for how mechanical thrombus integrity might be maintained during remodeling and vascular repair.

PIP2-induced membrane binding of the vinculin tail competes with its other binding partners.

Vinculin plays a key role during the first phase of focal adhesion formation and interacts with the plasma membrane through specific binding of its tail domain to the lipid phosphatidylinositol 4,5-bisphosphate (PIP2). Our understanding of the PIP2-vinculin interaction has been hampered by contradictory biochemical and structural data. Here, we used a multiscale molecular dynamics simulation approach, in which unbiased coarse-grained molecular dynamics were used to generate starting structures for subsequent microsecond-long all-atom simulations. This allowed us to map the interaction of the vinculin tail with PIP2-enriched membranes in atomistic detail. In agreement with experimental data, we have shown that membrane binding is sterically incompatible with the intramolecular interaction between vinculin's head and tail domain. Our simulations further confirmed biochemical and structural results, which identified two positively charged surfaces, the basic collar and the basic ladder, as the main PIP2 interaction sites. By introducing a valency-disaggregated binding network analysis, we were able to map the protein-lipid interactions in unprecedented detail. In contrast to the basic collar, in which PIP2 is specifically recognized by an up to hexavalent binding pocket, the basic ladder forms a series of low-valency binding sites. Importantly, many of these PIP2 binding residues are also involved in maintaining vinculin in a closed, autoinhibited conformation. These findings led us to propose a molecular mechanism for the coupling between vinculin activation and membrane binding. Finally, our refined binding site suggests an allosteric relationship between PIP2 and F-actin binding that disfavors simultaneous interaction with both ligands, despite nonoverlapping binding sites.

Site-Specifically-Labeled Antibodies for Super-Resolution Microscopy Reveal In Situ Linkage Errors.

The precise spatial localization of proteins in situ by super-resolution microscopy (SRM) demands their targeted labeling. Positioning reporter molecules as close as possible to the target remains a challenge in primary cells or tissues from patients that cannot be easily genetically modified. Indirect immunolabeling introduces relatively large linkage errors, whereas site-specific and stoichiometric labeling of primary antibodies relies on elaborate chemistries. In this study, we developed a simple two-step protocol to site-specifically attach reporters such as fluorophores or DNA handles to several immunoglobulin G (IgG) antibodies from different animal species and benchmarked the performance of these conjugates for 3D STORM (stochastic optical reconstruction microscopy) and DNA-PAINT (point accumulation in nanoscale topography). Glutamine labeling was restricted to two sites per IgG and saturable by exploiting microbial transglutaminase after removal of N-linked glycans. Precision measurements of 3D microtubule labeling shell dimensions in cell lines and human platelets showed that linkage errors from primary and secondary antibodies did not add up. Monte Carlo simulations of a geometric microtubule-IgG model were in quantitative agreement with STORM results. The simulations revealed that the flexible hinge between Fab and Fc segments effectively randomized the direction of the secondary antibody, while the restricted binding orientation of the primary antibody's Fab fragment accounted for most of the systematic offset between the reporter and α-tubulin. DNA-PAINT surprisingly yielded larger linkage errors than STORM, indicating unphysiological conformations of DNA-labeled IgGs. In summary, our cost-effective protocol for generating well-characterized primary IgG conjugates offers an easy route to precise SRM measurements in arbitrary fixed samples.