IIbβ3 on discoid diabetic platelets, increasing its association rate with immobilized fibrinogen. This compressive force-induced integrin activation is calcium and PI 3-kinase dependent, resulting in enhanced integrin affinity maturation and exaggerated shear-dependent platelet adhesion. Analysis of discoid platelet aggregation in the mesenteric circulation of mice confirmed that diabetes leads to a marked enhancement in the formation and stability of discoid platelet aggregates, via a mechanism that is not inhibited by therapeutic doses of aspirin and clopidogrel, but is eliminated by PI 3-kinase inhibition. These studies demonstrate the existence of a compression force sensing mechanism linked to αIIbβ3 adhesive function that leads to a distinct prothrombotic phenotype in diabetes.Diabetes is associated with an exaggerated platelet thrombotic response at sites of vascular injury. Biomechanical forces regulate platelet activation, although the impact of diabetes on this process remains ill-defined. Using a biomembrane force probe (BFP), we demonstrate that compressive force activates integrin α IIbβ3 on discoid diabetic platelets and that platelet aggregates can be eliminated by PI 3-kinase inhibition, but not by anti-thrombotics aspirin or clopidogrel.Diabetes is associated with an increased thrombotic response, but the mechanism is unknown. Here the authors demonstrate that compressive force activates integrin α Most individuals with diabetes die from the complications of cardiovascular diseases, particularly the acute coronary syndromes. Individuals with diabetes develop more widespread and advanced atherosclerotic lesions, and these plaques are more prone to rupture compared to non-diabetic individuals. Moreover, the thrombotic response at sites of atherosclerotic plaque rupture is typically exaggerated in diabetes, increasing the risk of vaso-occlusive thrombus formation, myocardial infarction and sudden death.Diabetes mellitus is becoming one of the major threats to human health and longevity in the 21st century. Based on current trends, children born after the year 2000 will have up to a 30% life-time risk of developing diabetes, leading to a 20–30% reduction in life expectancy2. Platelets from individuals with diabetes are more reactive than platelets from non-diabetics, as evidenced by an increased response to soluble agonist stimulation5 along with enhanced adhesion and aggregation responses on thrombogenic surfaces7. They are also more effective at supporting blood coagulation and thrombin generation8. The mechanisms regulating platelet reactivity in diabetic patients are complex and not completely understood. Following stimulation, platelets from diabetic patients have elevated levels of cytosolic calcium9 and generate higher levels of thromboxane A2 (TxA2)11. Chronic hyperglycemia leads to non-enzymatic glycation of platelet membrane proteins13 and upregulation in the function of the platelet P2Y12 receptor15. Reduced intracellular levels of antioxidants16, enhanced formation of soluble advanced glycation end products (AGEs)17, oxidative inactivation of the SERCA2 Ca-ATPase18, as well as mitochondrial dysfunction contribute to alterations in platelet reactivity in diabetics20.Platelets play a central role in the development of coronary disease by initiating and propagating plaque development, as well as promoting thrombus formation on the surface of disrupted plaques21. Despite the introduction of more potent P2Y12 antagonists, such as ticagrelor, diabetes remains associated with an elevated incidence of thromboembolic complications. Interestingly, integrin αIIbβ3 antagonists, the most potent class of antiplatelet agents, appear to work most effectively in diabetics23, indicating that dysregulation of integrin αIIbβ3 function is likely to be an important process underlying the diabetic prothrombotic phenotype.The clinical management of thrombosis risk for individuals with diabetes is complicated by the fact that platelets from diabetics are less responsive to the platelet inhibitory effects of the conventional antiplatelet agents, aspirin, and clopidogrelIIbβ3 activation and the kinetics of thrombus growth remains ill-defined24. This is likely to be clinically important as diabetics are more likely to form stable vaso-occlusive thrombi that precipitate organ injury25. Experimental studies have demonstrated that the efficiency of thrombus growth in vivo is influenced by the interplay of two distinct, but complementary, platelet aggregation mechanisms26. The first involves a rheology-dependent  platelet aggregation mechanism that is primarily mediated by discoid platelets. This mechanism is important for the initial recruitment of platelets to sites of vascular injury, particularly under conditions of disturbed blood flow27. The second is a soluble agonist-dependent aggregation mechanism that stabilizes formed aggregates. The biomechanical platelet aggregation mechanism primarily involves discoid platelets in a low-activation state. Aggregation of these platelets is initiated by hemodynamic shear gradients and requires the co-operative adhesive function of the platelet receptors GPIb and integrin αIIbβ327. The second aggregation mechanism involves agonist-induced platelet activation that primarily serves to upregulate integrin αIIbβ3 adhesive function and stabilize platelet aggregates. As a consequence, developing thrombi exhibit a heterogeneous structure of platelets in various degrees of activation and stability, ranging from fully activated and degranulated platelets in the stable thrombus ‘core’, to minimally activated, weakly adherent discoid platelets in the dynamic thrombus outer ‘shell’28.The manner in which diabetes effects αIIbβ3 activation, leading to a shear and red blood cell (RBC)-dependent increase in discoid platelet adhesion and aggregation in vitro and in vivo. Biomembrane force probe (BFP) analysis of platelet adhesion receptor–ligand kinetics at a single-platelet level indicates that in diabetics, increased sensitivity of platelets to shear forces is due in part to dysregulated integrin αIIbβ3 compression force sensing.In this report, we have examined the impact of chronic hyperglycemia on platelet responses to biomechanical and agonist stimulation using a streptozotocin (STZ) murine model of diabetes. Surprisingly, chronic hyperglycemia for up to 10 weeks in the mouse, did not result in increased platelet sensitivity to soluble agonist stimulation in vitro and in vivo. In contrast, chronic hyperglycemia resulted in an enhancement in biomechanical α30. This model allows precise control over the onset of hyperglycemia and enables assessment of platelet function independent of the confounding effects of other cardiovascular risk factors or overt vascular disease31. Hyperglycemia (blood glucose levels of >15 mM) was confirmed 1 week post streptozotocin injection and in vivo thrombosis studies were performed on mice 7–10 weeks post streptozotocin injection. In initial studies, we investigated the impact of disturbed blood flow on the platelet aggregation response in diabetic mice using a needle in situ model of platelet thrombus formation33. In this model, the tip of a microinjector needle is inserted into the center of the lumen of a mesenteric vessel, creating a local region of flow disturbance , completely inhibited discoid platelet aggregation around the needle tip in both diabetic and non-diabetic mice -induced mouse model of type 1 diabetesnce Fig. . Discoidnce Fig. . Platelence Fig. , 0–120 snce Fig. . These pnce Fig. . Analysince Fig. . A similnce Fig. . In cont27. Human diabetic platelets have enhanced responsiveness to soluble agonist stimulation, manifesting as increased rates of platelet aggregation at threshold levels of platelet stimulation35. To investigate whether mouse diabetic platelets had increased responsiveness to soluble agonist stimulation in vivo, we utilized an endothelial needle injury model of thrombosis, which perturbs, but does not denude, the endothelium at the site of injury and enables the local microinjection of soluble agonists at sites of thrombus formation36. Platelet activation in this model, prior to the injection of soluble agonists, is critically dependent on the generation of thrombin on the perturbed endothelium37. Interestingly, the initial recruitment and formation of platelet aggregates in this model were no different between control and diabetic mice  was dysregulated by diabetes, whole blood was perfused over FGN matrices at both venous (600 s−1) and arterial (1200 s−1) shear rates. The number of diabetic platelets adherent to FGN was increased 2–3-fold relative to non-diabetic platelets  over an immobilized fibrinogen matrix resulted in a twofold increase in platelet adhesion relative to non-diabetic healthy donors 45, although how compression forces influence platelet adhesive function remains largely unknown. To address this, we developed a novel compression assay using a biomembrane force probe (BFP). The probe bead was functionalized with FGN . If a bond connects the bead and platelet during their separation, the RBC would be stretched and report a tensile force  reflect the likelihood to find a receptor–ligand bond or bonds at the time of detection. To examine RBC-platelet collision effect, we measured the platelet adhesion frequencies with various compressive forces ranging from 5 to 40 pN. Interestingly, the adhesion frequency Pa was found to increase with the compressive force fc  reconstitution assays we made the surprising observation that the shear-dependent increase in diabetic platelet adhesion was RBC-dependent. Perfusion of isolated mouse platelets over FGN in the absence of RBCs yielded comparable numbers of adherent diabetic and non-diabetic platelets Fig. . After rFGN Fig.  and drivrce Fig. . The int fc Fig. . This ef fc Fig. . Moreove fc Fig. , suggestfc, and reaching a maximum (twofold increase) at saturating forces of fc > 20 pN  were measured over a range of contact times (tc) and various compression forces  were isolated and the adhesion frequency examined in an identical fashion using the BFP assay (fc = 20 pN). In accordance with results obtained from mouse platelets, FGN binding was enhanced in diabetic human platelets  with more recent onset of diabetes and therefore less likelihood of overt vascular disease. Similar to adults with type 1 diabetes, this pediatric group also exhibited enhanced platelet adhesion frequency to FGN by at fc = 20 pN  in the absence of force using flow cytometry50 , exerted via the FGN-bearing probe induced much higher level of JON/A binding to diabetic than control platelets . The FGN adhesion frequency to non-diabetic platelets fluctuated around the baseline, probably due to a small number of stochastic events. In sharp contrast, the initially slightly higher FGN adhesion frequency to diabetic platelets increased by ~2-fold within the first 125 s and by another 50% in the following 125 s  on the BFP at 5 s Fig. . As expe5 s Fig. , thus co5 s Fig. . These oIIbβ3 on-rate enhancement by compressive force in diabetic platelets is due to a biological rather than biophysical mechanism . This assertion was also supported by the observation that the frequency of fibrinogen adhesion to purified integrin αIIbβ3 coated on a glass bead was not enhanced with increasing contact force , especially the type 1 PI3Kβ isoform, in regulating integrin α0 s Fig. . Similar0 s Fig. . Importa0 s Fig. . These f−/−) mice rendered diabetic by streptozotocin injection  in diabetic mice Fig. , with thIIbβ3 senses and responds to mechanical stimulation on platelets remains unclear, but is likely to be a fundamental issue in platelet biology given the central role played by this receptor in mediating platelet-endothelial and platelet–platelet interactions under the influence of hemodynamic forces. The studies presented here demonstrate the existence of a compression force sensing mechanism utilized by platelets to regulate the adhesive function of integrin αIIbβ3. This mechanosensing mechanism is operative in discoid platelets, triggered by a threshold level of applied compressive force, and requires intracellular signaling events linked to the activation of integrin αIIbβ3. Functionally, it is associated with the enhanced adhesion of discoid platelets to immobilized fibrinogen and vWF, and an exaggerated discoid platelet aggregation response in vivo. In contrast, agonist-induced increases in integrin αIIbβ3 affinity were no different between diabetic and non-diabetic mouse platelets. This surprising finding was mirrored in vivo, where microinjection of either ADP or thrombin was not associated with an exaggerated thrombotic response in diabetic mice. Significantly, dysregulated biomechanical αIIbβ3 activation is insensitive to the platelet inhibitory effects of aspirin and P2Y12 receptor antagonists, but is highly sensitive to inhibitors of αIIbβ3 outside-in signaling, such as PI 3-kinase. These findings define a distinct diabetic prothrombotic mechanism linked to dysregulated biomechanical integrin αIIbβ3 activation that may partly explain resistance to oral antiplatelet therapies.How integrin α45. Notably, most studies make a common assumption that adhesion receptors on the platelet surface do not undergo marked changes during the RBC-dependent platelet margination, which may not be the case. It is likely that RBCs increase platelet compression against ligand surfaces to enhance the formation of nascent adhesive contacts. However, the molecular insights underlying the RBC “compressive effects” have been difficult to study, largely due to the technical challenge of applying single-molecule nanotools to live cells as small and reactive as platelets. The recent advancements in the BFP assay have enabled investigation of the single-receptor mechanosensing behavior on the surface of live platelets56. Using the BFP, we have demonstrated that a threshold force is required to enhance αIIbβ3 engagement of immobilized fibrinogen and that the efficiency of adhesion was influenced by both the magnitude and repetitive nature of the applied compressive force.In contrast to tensile and shear forces, which are far more extensively studied in mechanobiology, the biological effects of compressive force and the mechanisms of its reception, transmission and transduction in cells is poorly defined. Our study has provided the first evidence demonstrating that compression force sensing by platelets alters the adhesive behavior of integrin receptors. Insight into this was first provided from our in vitro flow assays where a critical role for RBCs was identified to differentially enhance the adhesive phenotype of diabetic platelets. To date, most studies have suggested that the primary effect of RBCs is to increase platelet transport (margination) from the blood stream to the vessel wall, thereby enhancing the probability of platelets forming adhesive contacts45. However, direct measurement of compression force on a platelet in free flow is technically challenging. For a platelet tethered to a surface by an adhesive contact at the rear end, we have performed a calculation as previously published58. For the venous to arterial shear rates studied in this report, γ = 600, 1800, and 5000 s−1, we estimated the compression forces fc = 11–24, 35–73, and 98–202 pN, respectively, relevant to those tested in the BFP experiments (see Supplementary Note). However, the precise relationship between the compression forces applied in our BFP assays and those experienced by platelets in vivo will be an important question for future investigation.Our use of repetitive compression cycles in the BFP assays is a reductionist approach to model platelet translocation dynamics, wherein individual platelets continuously form and break bonds. It is likely that translocating platelets experience continuous compression forces due to frequent collisions with RBCs in the bulk flowIIbβ3 activation will also be an important question for future studies but we postulate multiple potential mechanisms. The first possibility is in accordance with the force-through-lipid model which has been established for mechanosensitive ion channels i.e., MscL59. In this scenario, compressive force normal to the membrane is converted into tension in the membrane, which may trigger the opening of Ca2+ ion channels and induce integrin activation. This is consistent with our observation that chelating extracellular calcium abolished αIIbβ3–FGN adhesion enhancement on diabetic platelets. The second possibility may be related to the force-through-filament principle as the compressive force is sensed by the cytoskeleton rather than plasma membrane. In response to the external compressive force, the platelet cytoskeleton might undergo local remodeling, leading to integrin activation. Indeed, compressive force has been shown to alter the growth of branched actin-filaments at the leading edge of crawling cells60. Recent experimental and modeling studies have shown that integrin inside-out activation requires the cytoskeletal force to be transmitted through talin to the cytoplasmic tail of the β subunit63. An additional possibility may be related to the glycocalyx, a layer of glycoprotein–polysaccharide complex on the cell surface, since a recent study has demonstrated that local compression of the glycocalyx near integrin adhesive contacts promotes integrin clustering and focal adhesion maturation64.How compressive forces exerted on the platelet membrane leads to integrin α31. Consistent with this, we have found dysregulated calcium flux and PI 3-kinase signaling in our STZ-diabetes mouse model, verifying previous findings30. The STZ-diabetes mouse model also exhibits other important diabetes-related pathogenic mechanisms, including increased oxidative stress and the generation of advanced glycation end products17. A previous study using the STZ model of diabetes has reported an enhanced platelet aggregatory response to low concentrations of ADP and PAR4-AP, in conjunction with augmented signaling processes downstream of their respective receptors30. The discrepancy between our findings and those by Stolla et al.30 are not immediately apparent, although presumably are related to methodological differences. The studies presented here were performed using mice with confirmed hyperglycemia of 7–10 weeks in duration, whereas the hyperglycaemic duration in studies by Stolla et al.30 was not specified. It is entirely possible that there may be mouse strain, as well as species-specific differences in platelet responsiveness to chronic hyperglycemia. It is also possible that exaggerated platelet responses in human platelets (particularly in type 2 diabetes) may occur at least in part as a result of additional cardiovascular risk factors, such as dyslipidemia and hypertension. An interesting possibility that warrants further investigation is whether the duration of hyperglycemia has a major influence on the sensitivity of platelets to mechanical or chemical stimuli. In this context, most studies in humans have employed subjects with chronic hyperglycemia over many months and years, so it will be important to investigate time-dependent changes in platelet reactivity soon after diabetes diagnosis, particularly in patients with type 1 diabetes. Nonetheless, our demonstration that dysregulated biomechanical integrin αIIbβ3 activation occurs in platelets from both STZ-treated mice and humans with type 1 diabetes, independent of other cardiovascular risk factors, suggests that this process is primarily induced by biochemical changes associated with chronic hyperglycemia.The demonstration that chronic hyperglycemia in the mouse primarily resulted in a normal platelet response to agonist stimulation was surprising, since many biochemical changes associated with human diabetes can be recapitulated in rodent diabetes models2 and ADP35 and collagen exposure34. This may partly explain the requirement for higher doses of aspirin or clopidogrel to reduce thrombosis risk in diabetes21. Our demonstration of dysregulated biomechanical integrin αIIbβ3 activation in diabetic platelets may have direct clinical relevance, since it may partly explain the effectiveness of integrin αIIbβ3 antagonists in diabetic individuals. Our findings from multiple distinct experimental approaches, including the calcium flux assay under shear, BFP time-lapse adhesion assay and FGN-to-JON/A ‘switch’ assay are consistent with the possibility that integrin downstream signaling is dysregulated in diabetic platelets, leading to an exaggerated discoid platelet adhesion response. In this regard, the lipid kinase PI3K, especially its β isoform, which is an important mediator of αIIbβ3 outside-in signaling has previously been demonstrated to play a key role in integrin αIIbβ3-dependent platelet adhesion53. Indeed, inhibition of PI3K markedly inhibits αIIbβ3 adhesion maturation and αIIbβ3-mediated platelet adhesion under shear. In contrast, PI3K does not appear to have a significant role in initiating αIIbβ3 activation following soluble agonist stimulation, but rather, sustains αIIbβ3 activation through regulation of Rap1b and AKT65. Furthermore, PI3K has been demonstrated in other cell types to play an important role in regulating the activation of integrin αvβ3 in response to biomechanical force66. Given the effectiveness of PI3K inhibition in preventing biomechanical platelet activation in diabetes, therapeutic targeting of integrin αIIbβ3 signaling pathways may represent an innovative approach to reduce the prothrombotic effects of diabetes.How compressive force sensitivity relates to the many aspects of platelet hyperactivity and dysfunction reported in diabetes remains an important issue for future investigation. Some of the platelet abnormalities in diabetes involve receptors or signaling pathways linked to platelet activation by soluble agonists, most notably TxA−/−) mice were obtained from the Jackson Laboratory .All procedures involving the use of mice for both in vitro and in vivo studies were approved by the Alfred Medical Research and Education Precinct (AMREP) Animal Ethics Committee  (project # E/1068/2011/M and E/0856/2009/M), and the University of Sydney Animal Ethics Committee (Project # 2014/620). All C57BL/6J male mice were sourced from AMREP animal services or Australian BioResources . The PI3Kβ-deficient (PI3Kβ−1) for 5 consecutive days67. Blood glucose levels were examined 1 week post STZ injection, and monitored during their maintenance and prior to experiments. Mice which had blood glucose levels >15 mM were considered diabetic. Diabetic mice 7–10 weeks post STZ injection were used in this study, unless indicated otherwise. Diabetes was also induced in 6-week-old male PI3Kβ−/− mice and hyperglycemia was confirmed using the same procedures.Diabetes was induced in 6-week-old male C57BL/6J mice via daily intraperitoneal injection of streptozotocin  with the approval of the Alfred Health Ethics Committee (project # 388/11 and 409/14). Age and gender matched non-diabetic healthy donors were also recruited as controls. All participants were confirmed devoid of any anti-platelet medication for the preceding 2 weeks, and were moreover free from any history of bleeding disorder, anemia, renal impairment, and acute coronary syndrome or coronary intervention (<30 days). Patients with type 2 diabetes were excluded from this study. The total number of participants enrolled in these studies included 24 diabetic patients  and 24 non-diabetic healthy donors .Juvenile type 1 diabetic patients were recruited under the approval of the Emory University Ethics Committee (Protocol number H14444 at Georgia Tech and IRB00077577 at Emory University) and the studies on these patients were conformed to the principles outlined in the Declaration of Helsinki. Blood was obtained from these patients in the Emory Children’s Center on the Emory University campus. All participants were aged between 7–19 years old, and did not have a history of bleeding disorder, anemia, renal impairment, and acute coronary syndrome or coronary intervention (<30 days). All participants and their accompanying guardians were provided with a verbal explanation of the study. A verbal agreement and a signed consent form were respectively collected from the participants and accompanying guardians, stating that they agree for blood donation, and for the publication of the data collected using their blood. The participants in this cohort included 3 non-DM individuals  and 5 DM patients . In all cases, no fasting was requested prior to blood donation.−1) for 2 h at room temperature, or overnight at 4 °C, then blocked with 20 mg mL−1 BSA for 30 min. Hirudinated mouse whole blood was perfused through the FGN coated microslides at 600 s−1 for 5 min. Platelet adhesion was monitored using an inverted Leica DMIRB microscope and differential interference contrast (DIC) microscopy . Platelet adhesion was recorded using Micro-manager v1.4 for off-line analysis. For perfusion studies with human whole blood, mouse washed platelets or reconstituted blood, platelets were perfused through FGN-coated microslides  at 200 or 300 s−1 for 5 min. For mouse blood perfusion studies using murine vWF matrix or spread platelet monolayers, polydimethylsiloxane (PDMS) microfluidic channels (0.3 × 0.1 mm (height)) were used68. To capture vWF, the bottom of PDMS device was coated with an anti-vWF antibody AB7356  for 1 h at room temperature, then incubated with mouse plasma for 2 h at room temperature to capture vWF69. After the BSA blockade for 0.5 h, the mouse blood was perfused through at preset shear rates . To mitigate the pre-activation effect of microfluidics on platelets, the hirudinated blood was pre-treated with amplification loop blockers (ALB): ADP receptor blockers MRS2179 (100 μM), 2-MeSAMP (10 μM), cyclooxygenase inhibitor indomethacin (10 μM).For perfusion studies using mouse whole blood, nitric acid-washed glass microslides (0.1 × 1.0 mm: height × width) were coated with fibrinogen . Platelets were considered adherent if they remained attached to the matrix for at least 2 s. Platelets which moved <1 cell diameter in 10 s following tethering to the matrix were classified as stationary cells, and platelets that rolled (continuously or stop/start) over a 10-s period were considered rolling cells.55. Its spring constant of kBFP = 0.15–0.3 pN nm−1 is determined by the aspiration pressure and the radii of the micropipette and the RBC-bead contact area70. The glass bead, also called “probe” bead, was coated with FGN, vWF-A1 or JON/A via maleimide-PEG3500-NHS . The other micropipette aspirates a platelet and dives the platelet to contact the probe under a defined compressive force (fc = 5–40 pN) for a certain contact time (tc)  to allow bond formation. The platelet is then retracted at a constant speed (3.3 μm s−1) for bond detection. During the retraction phase, a bond formation is signified by a tensile force. Both compressive and tensile forces are measured by quantifying the RBC deformation, which is detected by the axial movement of the probe bead tracked by a high-speed camera. Each aspirated platelet is subjected to repetitive cycles  of touch with the probe bead, and the fraction of bond formation over the cycles was determined as adhesion frequency Pa.The BFP utilizes two micropipettes, one aspirating a RBC with a glass bead attached on the apex via biotin-streptavidin interaction to serve as a force transducerPa depends on the contact time (tc). The 2D zero-force cellular on-rate (mrmlAckon) and off-rate (koff) can be derived by fitting the Pa vs. tc curve with the model Pa = 1 − exp{−mrm1Ackon[1 − exp(−kofftc)]/koff}, where Ac is the contact area, assumed to be constant for a given compression force. The site densities of integrin αIIbβ3 (mr) on mouse platelets and fibrinogen (ml) on beads were measured as mr = 681 – 2098 µm−2 for non-DMs and 969–2262 µm−2 for DMs and ml = 90 µm−2, using a flow cytometry method with the Quantum™ FITC-5 MESF  calibration beads from Bangs Laboratories 47. The signals were detected by the FITC labeled anti-αIIbβ3 mAb (Leo.F2) from Emfret Analytics  and the anti-fibrinogen γ chain mAb (LS-C418056) from LifeSpan BioSciences , respectively. Then we divided the measured cellular on-rate parameters by these site density numbers to obtain the effective 2D on-rates (Ackon). The effective 2D affinity can be calculated from AcKa = Ackon/koff.To assess the kinetics of the bond formation, the aspirated platelet was allowed to contact the ligand coated bead for various durations, since the adhesion frequency 49. Moreover, the 2D kinetic rates we measured are on the condition that the integrins are NOT subject to tensile force. Whereas the platelets were subject to various compression forces. The changing integrin affinity is a biological response of the platelet that involves signaling.Of note, for adhesion frequency analysis to extract 2D kinetics, we made no claim on whether the measurements were done under the conditions of single molecular interactions. Such claim is not needed because at higher adhesion frequencies the measurements were not made under single bond conditions and because such conditions are not needed for our conclusions on affinity change and on-rate changes−1) or ketamine/xylazine (150/15 mg kg−1). The anesthetized mice were provided with oxygen (0.5 L min−1) via a mask during the entire experimental procedure.C57BL/6J control or diabetic male mice with a body weight of 18–25 g were subjected to various in vivo thrombosis models. All mice were anesthetized with sodium pentobarbitone  with a micropipette puller  such that the needle tip had a diameter of 2–3 µm. The needle was inserted into the lumen of mesenteric vessels (100–200 µm in diameter), with the needle tip positioned ~20 µm from the base of the vessel using an Injectman micromanipulator . Thrombus formation at the needle tip was monitored for 4 min under real-time DIC microscopy using an Olympus IX81 inverted microscope with a ×40 objective, and recorded via EM-CCD camera  for off-line analysis using Visiview software 2.1.4 . Thrombus surface area at the indicated times was determined using ImageJ software. To examine platelet P-selectin expression during thrombus formation, mice were preadministered DyLight 649-anti-GPIbβ Ab (100 µg kg−1) and Alexa 546-anti P-selectin Ab (50 µg kg−1). P-selectin expression and thrombus formation were monitored using Nikon A1R confocal intravital microscopy with a ×40 objective. Fluorescent images were captured using NIS software, Version 4 . Where indicated, mice were pre-treated with the anti-platelet agents aspirin  and clopidogrel  or vehicle (Gum Arabic) 24 h and 2 h prior to experimentation. Mice were also treated with the PI3Kβ inhibitor TGX221 (2.5 mg kg−1) or vehicle (DMSO), 10 min prior to needle in situ thrombosis. To inhibit integrin αIIbβ3 ligand binding, mice were infused intravenously with integrilin (4 mg kg−1) via a carotid catheter every 15 min during the entire experimental period.Thrombi were induced in the mesenteric venules or arterioles of diabetic and non-diabetic mice using the needle in situ model37. Thrombi were created by repetitive puncture (×10) of the mesenteric venules using a needle that had a 2 µm tip and was controlled by an Injectman micromanipulator. Thrombus formation at sites of vessel injury was monitored using intravital DIC microscopy, and thrombus surface area determined using Image J. Where indicated, the thrombi were subjected to local microinjection of ADP (10 mM) or thrombin (100 U mL−1) by positioning the microinjector needle, ~40 µm upstream of the thrombi in the direction of blood flow. The agonist injection was carried out under a prespecified pressure (1000 kPa) and duration (1.5 s) for 10 times. Of note, the needle does not breach or denude the endothelium. The thrombin was only locally injected, 20–40 µm upstream of the thrombus, not to the entire venule, and the quantity injected was very small (0.14 nL). Local thrombin injection also served as a positive control for induction of P-selectin expression in the needle in situ thrombosis model presented in main Fig. Thrombi were induced at sites of mesenteric venule injury via needle puncture−1), Alexa-488 Annexin V (0.1 mg kg−1), GPIbβ Ab , or P-selectin Ab  to monitor endothelial cells, endothelial cell death, platelet adhesion, and activation, respectively. In order to examine ICAM-1-depedent fibrinogen deposition to the crushed endothelium, mice were pre-injected with Alexa-488-fibrinogen (17 mg kg−1) in the absence or presence of the ICAM-1 blocking Ab . To assess αIIbβ3-dependent platelet adhesion, mice were injected with integrilin (4 mg kg−1) every 15 min Following crush injury, DIC, fluorescence and confocal intravital microscopies were performed as described under the ‘needle in situ’ thrombosis model. For platelet adoptive transfer studies, platelets were isolated from non-diabetic and diabetic donor mice, labeled with Alexa 488-anti-GPIbβ Ab, and resuspended in Tyrode’s buffer at a concentration of 1 × 109 mL−1. The recipient non-diabetic mice were sytemically administered DyLight 649-anti-GPIbβ Ab (100 μg kg−1) to label endogenous platelets, and then infused with donor platelets via inferior vena cava to achieve a donor/recipient ratio of 1/10. The mesenteric venules of the recipient mice were then subjected to mechanical crush injury. The adhesion of infused non-DM, and DM donor platelets and recipient endogenous platelets was monitored using confocal intravital microscopy, and images taken pre- and 10 min post-injury. The fluorescence intensity of the circulating endogenous and infused donor platelets pre-crush, and those adherent to the endothelium post-crush were quantified using ImageJ, and expressed as the ratio of donor over recipient platelets.A microinjecter needle tip was blunted (30–50 μm) and positioned above the exposed mesenteric venule. The mesenteric venule was then crushed twice by the blunted needle using the Injectman micromanipulator. Platelet adhesion to the crushed endothelial area was monitored at real time using DIC microscopy. Where indicated, mice were systemically injected with anti-PECAM Ab (FITC, 0.2 mg kgThe data that support the findings of this study are available from the corresponding author upon reasonable request.Supplementary InformationDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2


Trypanosoma brucei and Leishmania mexicana. This showed quantitatively how chirality in T. brucei cell shape confers highly directional swimming. High speed videomicrographs showed that T. brucei, L. mexicana and a T. brucei RNAi morphology mutant have a range of shape asymmetries, from wild-type T. brucei  to L. mexicana . The chiral cells underwent longitudinal rotation while swimming, with more rapid longitudinal rotation correlating with swimming path directionality. Simulation indicated hydrodynamic drag on the chiral cell shape caused rotation, and the predicted geometry of the resulting swimming path matched the directionality of the observed swimming paths. This simulation of swimming path geometry showed that highly chiral cell shape is a robust mechanism through which microscale swimmers can achieve highly directional swimming at low Reynolds number. It is insensitive to random variation in shape or propulsion . Highly symmetric cell shape can give highly directional swimming but is at risk of giving futile circular swimming paths in the presence of biological noise. This suggests the chiral T. brucei cell shape  may be an adaptation associated with the bloodstream-inhabiting lifestyle of this parasite for robust highly directional swimming. It also provides a plausible general explanation for why swimming cells tend to have strong asymmetries in cell shape or propulsion.Swimming cells typically move along a helical path or undergo longitudinal rotation as they swim, arising from chiral asymmetry in hydrodynamic drag or propulsion bending the swimming path into a helix. Helical paths are beneficial for some forms of chemotaxis, but why asymmetric shape is so prevalent when a symmetric shape would also allow highly directional swimming is unclear. Here, I analyse the swimming of the insect life cycle stages of two human parasites;  Trypanosoma brucei (which causes sleeping sickness/trypanosomiasis) and Leishmania mexicana (which causes leishmaniasis). Using new computational methods to determine the three dimensional shape of swimming cells I showed that T. brucei have a helical shape which causes rotation as the cell swims, and the geometry of the resulting swimming path makes the cell movement highly directional. In contrast, L. mexicana are symmetrical, do not rotate, and their swimming paths are curved and have low directionality. Using a T. brucei mutant I showed that the cell structure responsible for the helical shape while swimming is the flagellum attachment zone. This explains a key function of this structure. Finally, simulations showed the phenomenon of rotation while swimming is a way cells can ensure highly directional swimming along a controlled helical path, overcoming random variation in cell shape or propulsion. This provides a general explanation for why swimming cells are often asymmetric and tend to follow helical paths.Swimming cells often follow a helical swimming path, however the advantage of helical paths over a simple straight line path is not clear. To analyse this phenomenon, I analysed the swimming of the human parasites  Ploesoma[Paramecium[Caulobacter[Volvox[Chlamydomonas[Bacillus subtilis[Many swimming microorganisms and cells swim along helical paths; examples come from all scales of microscopic life, including the multicellular zooplankton Ploesoma, ciliatearamecium, and baculobacter. This haulobacter, and a hulobacter,5. Heliculobacter,5 and isulobacter,7. In ther[Volvox, the unimydomonas and the  subtilis. This be subtilis, but is Trypanosoma brucei (which causes sleeping sickness/African trypanosomiasis) is a characteristic example of this. It has a strongly asymmetric cell organisation with its single flagellum laterally attached to the cell body for much of its length by the flagellum attachment zone (FAZ)[Swimming along a straight line is a beneficial behaviour for chemotaxis, either to maximise displacement during run and tumble chemotaxis or to travel directly up a chemoattractant gradient in helical clinotaxis. The naïve approach to achieving straight line swimming is perfect symmetry in propulsion and cell shape, as an asymmetry in either could bend a straight swimming path into a futile circular path. However, cells which can achieve highly directional, near straight line, swimming tend to have chirally asymmetric propulsion or cell shape–3,8–10. one (FAZ),12, and one (FAZ)–17. Motione (FAZ),19, and one (FAZ),20. The one (FAZ), but notT. brucei is a member of a family of parasites which also includes Leishmania mexicana . T. brucei and L. mexicana share the typical features of trypanosomatid cells; a single flagellum and long, thin, cell body whose shape is defined by a parallel array of sub-pellicular microtubules[T. brucei and L. mexicana have differing asymmetry in cell shape; Leishmania morphogenesis and cell shape are more symmetric[rotubules. They acrotubules. Their mrotubules–25 and protubules–28. T. bsymmetric,29. All symmetric,16,30,31symmetric–34.T. brucei morphological mutant with a shorter FAZ[T. brucei cells have a helical shape (distorted by flagellum movement) while L. mexicana do not. The capacity for longitudinal rotation correlated with calculated torque from hydrodynamic drag under constant fluid flow due to helicity of cell shape. The T. brucei morphological mutant showed helical cell shape is conferred by the lateral attachment of the T. brucei to the cell body, suggesting the function of the extended flagellum attachment zone (FAZ) may be to generate helicity in cell shape and confer the ability for more robust directional swimming. As this extended FAZ is an adaptation associated with the bloodstream-inhabiting lifestyle of Trypanosoma parasites[Through analysis of these two human parasites, including a orter FAZ, I showeparasites, this maT. brucei arose from a long pitch, low radius, helical path. This simulation also quantitatively demonstrated that this spin stabilised swimming is tolerant of small variations in either shape or propulsion . However, cells which achieve highly directional swimming through avoiding asymmetry are not. This suggests that the commonality of chiral asymmetric shape and propulsion in swimming cells and microorganisms is to allow highly directional swimming in the presence of biological noise in cell shape and propulsion.Simulation of cell swimming with longitudinal rotation confirmed that rotation rate is sufficient to explain the large-scale swimming behaviours: ‘Spin stabilisation’ arose through the helical geometry of the resulting swimming path, and straight line swimming of Leishmania mexicana promastigote and Trypanosoma brucei procyclic trypomastigote are of similar size, both normally inhabit a similar (fly gut) environment, and both are grown at the same temperature (28°C). However, they have differences in morphology. Additionally, a viable morphological mutant of T. brucei procyclic trypomastigotes  has recently been described with a shortened FAZ which gives an intermediate (epimastigote-like) morphology[The orphology. This alL. mexicana and procyclic form (trypomastigote) T. brucei differ, their swimming was analysed using low magnification dark field videomicrographs. Videomicrographs were captured from parasites swimming in 0.25 mm deep chambers to minimise any surface entrainment effect. Cells were detected and swimming tracks were determined using automated tools written in the ImageJ macro language[L. mexicana cells adhered to a glass slide (instead of being free to swim). Cells could be reliably detected by this method within approximately 10 μm of the focal plane, with the proportion detected dropping exponentially beyond that range  and directional swimming . There was one class of behaviour for L. mexicana promastigotes; swimming  , whiirection . T. brucdirection,14,37. TT. brucei). Two stonality) . Within Given these differences in swimming I next considered two hypotheses as to how the differences may arise: (i) From the differences between the promastigote and trypomastigote morphology, or (ii) from differences in the flagellum beat structure and regulation between these two species.T. brucei procyclic trypomastigote morphology to increase similarity to the promastigote may alter the swimming behaviours to be more similar to the promastigote. This was tested using the T. brucei tetracycline inducible ClpGM6 RNAi cell line[If the cell morphology is responsible for the differences in swimming behaviours then alteration of cell line. The trycell line, althougcell line) grow mocell line.T. brucei (p>0.1) or average directional persistence , however there was a significant change  in directional persistence distribution . The flagellum beat was similar, with a canonical sinusoid-like shape in both cell types, but a larger amplitude in the epimastigote-like  but L. ote-like . When thote-like . Equivalote-like . TherefoT. brucei and L. mexicana promastigotes was occurrence of longitudinal rotation. Using high magnification high frame rate videomicrographs, rotation of trypomastigote (uninduced ClpGM6 RNAi), epimastigote-like (72 h ClpGM6 RNAi) and promastigote (L. mexicana) cells while swimming was analysed. Rotation was identified from changes in orientation of the posterior of the cell each time a new flagellum waveform initiated from the anterior flagellum tip. Number of cell rotations or flagellar beats over the length of the videomicrograph was used to calculate beat and rotation frequency. The reorientation of the cell posterior of the trypomastigote did not appear oscillatory, unlike previously described for the bihelical beat model of procyclic form motility[L. mexicana did not rotate around their longitudinal axis on this time scale (n = 6), while both induced and uninduced ClpGM6 RNAi did (n = 8 and n = 7 respectively). Rate of longitudinal rotation was significantly reduced  following ClpGM6 RNAi (The striking difference in cell movement while swimming between epimastigote-like  motility. The freGM6 RNAi . The coaκ(s), and torsion, τ(s), of the path as functions of displacement, s. For a cell undergoing an unchanging swimming behaviour (omitting Brownian motion and noise through variation of propulsion or cell shape) curvature and torsion are constant. It therefore follows a helical path with constant radius, r = κ/(κ2 + τ2), and pitch, h = 2πτ/(κ2 + τ2)[ωτ perpendicular to the direction of travel and torsion as a constant angular velocity ωκ parallel to the direction of travel respectively (corresponding to rotation of the Frenet-Serret frame)[The effect of longitudinal rotation rate on swimming behaviours can be mathematically predicted from the helical path shape generated by different longitudinal rotation rates,5. The s(κ2 + τ2). This caet frame). At low et frame), so the ωτ = 0 to 5 rad/s and ωκ = 0 to 5 rad/s at 5±2 μm/s including Brownian motion  and T. brucei epimastigote-like procyclic forms (ωτ = 2.5±0.7 rad/s)  . ωτ = 0.5±2 μm/s . A projeT. brucei and L. mexicana swimming, which allowed direct comparison to the T. brucei and L. mexicana swimming data  of promastigote L. mexicana gave a reduced capacity for highly directional swimming  were simulated using these angular velocity parameters. From these swimming paths simulated images, imitating low magnification videomicrographs, were generated . These sata Figs  and 2. Tswimming , also siswimming . The swiGM6 RNAi . This coT. brucei cells undergo complex movement as they swim due to the lateral attachment of the flagellum to the cell body by the FAZ. The cell presumably has an intrinsic shape defined by the organisation of sub-pellicular microtubule cytoskeleton[T. brucei trypomastigote and epimastigote-like cells, an analytical model of the cell movement was fitted to high frame rate videomicrographs to infer the effective shape of the cell exposed to hydrodynamic drag while the cell is swimming. This is the shape of the cell with the repetitive, cyclic, movement directly due to the flagellum beat subtracted, and is different from the shape the cell would attain if the flagellum was straight and not beating or frozen at a particular stage of the beat. It includes the averaged contribution of flagellar movement on cell shape, and a tumbling cell may not have the same shape. Note that this is not a physically accurate model; instead it is a numerical description of cell shape changes as the flagellum beats which can then be used to infer three dimensional cell shape from the two dimensional source data.oskeleton, howeverThe model comprises two parts: (i) A lateral displacement arising from the cell shape as it rotates longitudinally, and (ii) a lateral displacement arising from a planar sinusoid-like flagellar beat see , S3 Fig.n = 8) and epimastigote-like (n = 7) cells was calculated assuming a fluid flow of 1 μm/s. This confirmed the most helical/chiral regions contribute most torque . A simpler simulation based on the effective hydrodynamic shape of the cells was therefore used, treating the cell as a series of cylinders of different widths. Hydrodynamic drag on a cylinder at an angle relative to oncoming fluid flow generates a force orthogonal to the fluid flow which, if offset from the midline of the cell, causes a torque. The torque generated from drag on the effective hydrodynamic shape of trypomastigote . Low curvature, low torsion paths were sensitive to cell intrinsic noise, with cell to cell or time dependent random change in curvature (modelling morphogenetic noise and random variation in the flagellar beat respectively) reducing directionality. This curved the swimming path giving rise to a futile circular trajectory . Secondchia coli,44 and mchia coli). Determchia coli. The mecT. brucei use a canonical example of helical cell shape to cause rapid longitudinal rotation and highly directional swimming, while L. mexicana do not. Simulation of the resulting swimming behaviours reveals a possible explanation for why swimming microorganisms often have asymmetric shape, as the longitudinal rotation caused by asymmetric drag or propulsion leads to directional swimming insensitive to noise in shape or propulsion, while a highly symmetric shape is sensitive to such noise. This provides a quantitative framework for understanding the diverse, complex and asymmetric shapes of swimming cells and microorganisms seen across the scales of microscopic life [T. brucei and L. mexicana parasite swimming.By providing quantitative analysis of the phenomenon of ‘spin stabilisation’ using two human parasites, this work revealed pic life –3,8–10. 5 and 2×107 cells/ml by regular subculture, and cultures between 5×106 and 1×107 cells/ml were used for analysis. Culture density was measured with a CASY model TT cell counter (Roche Diagnostics).Leishmania mexicana (WHO strain MNYC/BZ/62/M379) were grown in M199 supplemented with 10% FCS and 50 mM HEPES·HCl pH 7.4. Procyclic form Trypanosoma brucei (strain Lister 427) and the inducible ClpGM6 RNAi cell line were groLow magnification videomicrographs for motility analysis were captured using dark field illumination at a frame rate of 5 Hz, using a 10× NA 0.25 objective on a DM5500 upright microscope (Leica Microsystems) and an Orca CCD camera (Hamamatsu Photonics) or Neo sCMOS (Andor) with an exposure time of 2 ms. 5 μl cell culture was held in a sealed 10×10×0.25 mm volume generated with an adhesive geneframe (Fischer Scientific), a slide and a coverslip, and imaged at the ambient temperature of 26°C. Cell swimming tracks from low magnification videomicrographs and simulated low magnification videomicrographs were analysed using custom automated analysis tools written in the ImageJ macro laT. brucei[δt for tracks at least 5 s long. Mean speed s = |vt| and directional persistence δt = 2 s to assay cell swimming behaviours and omit reciprocating movement directly due to the oscillation of the flagellar beat or cell rotation was selected by determining the population mean of autocorrelation directional persistence  for autocorrelation evaluation intervals between 0 and 10 s  and a Neo cooled sCMOS camera (Andor) with an exposure time of 5 ms. 1 μl cell culture was held in an area of approximately 10×10 mm, giving a volume depth of approximately 10 μm, and imaged at the ambient temperature of 26°C. Individual cells were cropped from the video, and swimming translation digitally subtracted assuming a constant velocity.p by x = r cos p, y = r sin p, z = cp giving a helix radius r = κ/(κ2 + τ2) and pitch h = 2πc = 2πτ/(κ2 + τ2). The length along a helical arc from p = 0 to p = P is given by P = 2π giving p = 0 and p = P.δx = r cos P − r, δy = r sin P, δz = cP, therefore displacement T, r and h allows the calculation of the ratio s/l as a measure of directionality from κ and τ. In the case of large P, where hP ≫ r, lateral displacement δx and δy become negligible and s ≈ cP. In this case Directionality of swimming omitting Brownian motion and biological noise was calculated assuming constant swimming speed and constant curvature and torsion giving a helical path. A helix can be defined parametrically with parameter s, longitudinal angular velocity ωτ and orthogonal rotational velocity ωκ, each randomised from a normal distribution with the specified standard deviation around the specified mean. Changes to cell location (vector l) and orientation  by cell movement was simulated in time steps (δt) of 200 ms using Euler’s method and calculating rotations using quaternions: First the cell was translated by sδt in the direction t, secondly t and n were rotated around b by ωκδt, thirdly b and n were rotated around t by ωτδt, then finally the cell was translated along t, n and b and rotated around t, n and b by randomised values to simulate Brownian motion.Directionality of swimming with Brownian motion and biological noise was calculated by numerical simulation . Each ceD = kT/6πrμ where k is the Boltzmann constant and T is the thermodynamic temperature . The cell was modelled as a prolate spheroid rd = 1.5 μm for displacement along t and rnp = 8.0 μm for displacement along n and b. For each time step rotational Brownian motion was simulated as normally distributed random rotation E = kT/8πμa3 where f of a prolate spheroid[fd) and minor axes (fnp) are given by:For each time step translational Brownian motion was simulated as normally distributed random displacement  relation D = kT/6 relation E = kT/8 spheroid, relativl|/st) was calculated.For calculation of swimming tolerance to sources of noise, swimming was simulated for 10,000 cells starting at the origin  and pr(z) are the amplitude and phase of cell displacement along the cell. ωr is the angular velocity.Displacement at any point along the cell body due to its rotation of an underlying shape is given by:Ab(z) and pb(z) are the amplitude and phase of movement due to the flagellar beat along the cell. ωb = 2πfb where fb is the frequency of the flagellar beat. Non-linear pb(z) allows modelling of both travelling flagellum beat waves and rigid cell oscillations due to the flagellar beat.Cell and flagellum displacement due the flagellar beat were approximated as a sinusoid:b(z) depends on the orientation of the cell at that point along its length, given by pr(z). In addition, the orientation of the flagellum may vary relative to the orientation of the cell. The combination of displacement due to cell body rotation and movement due to the flagellar beat is therefore:po(z) is a function of z describing the orientation at which the flagellum beat is oriented relative to the orientation of the cell pr(z).The flagellar beat is planar, therefore the observed magnitude of xc was manually fitted to high frame rate videomicrograph kymographs at 8 values of z to estimate the Ar(z), pr(z), Ab(z), pb(z) and po(z) functions, along with values of ωr and fb. Multiple values of pr, pb and po which fit the kymograph equally well at each z occur at intervals of 2π, therefore accurate fitting of the kymographs for intermediate z values by linear interpolation of the Ar(z), pr(z), Ab(z), pb(z) and po(z) was used to determine their correct value. Efforts to fit cell movement to an oscillating cell orientation, implied by a bihelical model of cell movement[ωr, were not successful. movement, insteadxc and yc share parameters, therefore fitting of xc to experimental data defines yc. Combined with an estimate of cell width w(z) this defines a three dimensional description of cell shape as it moves to swim. To confirm a good fit this was plotted as a phase contrast-like image in which defocus ) is represented as a colour shift from black (in focus) through white to grey.Ab(z) was set to zero for all z. The resulting three dimensional shape was rendered in Blender.To determine the effective hydrodynamic shape of the cell, Torque arising from hydrodynamic drag was calculated from theory of slender bodies in a Stokes flow,68. TorqS1 Fig(A) Correlation of the number of cells detected by the automated image analysis tools from a low magnification dark field micrograph and defocus distance for live L. mexicana cells adhered to a slide. (B) Distribution of cell path lengths generated by the automated image analysis tools from a dark field videomicrograph captured at 5 Hz of a sample of swimming L. mexicana. (C) Autocorrelation of directional persistence for L. mexicana and T. brucei. Detail of autocorrelation in the first 1 s is inset on a different vertical scale. Decay of autocorrelation fell sharply over the first 0.5 s (for T. brucei) and 0.1 s (for L. mexicana) and began a steady exponential decay for both species after 1 s. An evaluation interval of 2 s was therefore used for all analysis of cell swimming paths.(TIF)Click here for additional data file.S2 Fig(A) Diagram of the vectors l, t, n and b used to define cell location and orientation, the angular velocities ωκ and ωτ responsible for change in cell orientation and the speed s responsible for cell translocation as used for simulation of swimming. The swimming behaviours are derived from only these three parameters, and is not a hydrodynamic simulation based on cell shape. (B) Example three dimensional rendering of simulated swimming paths followed by cells undergoing promastigote swimming behaviours in a three dimensional volume. (C) Point spread function used to translate the three dimensional cell swimming paths into a simulated low magnification videomicrograph for comparison of swimming simulation to experimental data, and an example frame generated from the swimming paths shown in (B).(TIF)Click here for additional data file.S3 Fig(A) Diagram representing the analysis scheme used to infer three dimensional cell shape from observations in a single focal plane.  Examples illustrating the contribution of different components of cell movement to the fitting of xc to kymographs from uninduced (B) and 72 h induced (C) ClpGM6 RNAi T. brucei. xr is the contribution of cell shape and rotation, b is the contribution of the flagellar beat, xb = b sin(pr(z) + po(z) + ωrt) is the contribution of the flagellar beat taking into account orientation of the flagellar beat plane and xc = xr + xb is the combination of both cell body rotation and flagellum beating.(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S1 Video(top) trypomastigote cells (uninduced ClpGM6 RNAi T. brucei), (middle) epimastigote-like cells (72 h induced ClpGM6 RNAi T. brucei) and (bottom) a promastigote cell (L. mexicana). Playback is at ⅛ original speed.The swimming motion of (AVI)Click here for additional data file.


CHA1. To assess the generality of the observed function of Clp1 in transcription, we tested the effect of Clp1 on transcription on a genomewide scale using the Global Run-On-Seq (GRO-Seq) approach. GRO-Seq analysis showed the polymerase reading through the termination signal in the downstream region of highly transcribed genes in a temperature-sensitive mutant of Clp1 at elevated temperature. No such terminator readthrough was observed in the mutant at the permissive temperature. The poly(A)-independent termination of transcription of snoRNAs, however, remained unaffected in the absence of Clp1 activity. These results strongly suggest a role for Clp1 in poly(A)-coupled termination of transcription. Furthermore, the density of antisense transcribing polymerase upstream of the promoter region exhibited an increase in the absence of Clp1 activity, thus implicating Clp1 in promoter directionality. The overall conclusion of these results is that Clp1 plays a general role in poly(A)-coupled termination of RNAPII transcription and in enhancing promoter directionality in budding yeast.In budding yeast, the 3′ end processing of mRNA and the coupled termination of transcription by RNAPII requires the CF IA complex. We have earlier demonstrated a role for the Clp1 subunit of this complex in termination and promoter-associated transcription of  The complex has been implicated in cleavage-polyadenylation and coupled termination of transcription4. The termination function of the CF IA complex was demonstrated using a nuclear run-on assay that revealed the readthrough of RNAPII beyond the poly(A) site in the mutants of all four CF IA subunits5. The transcription readthrough phenotype, however, was demonstrated for just a few selected yeast genes. ChIP analysis identified that the whole CF IA complex is localized at the 3′ end of selected genes in accordance with its role in 3′ end processing and termination of transcription6. Genomewide analysis found Pcf11 crosslinked to the 3′ end of a majority of transcriptionally active yeast genes7. These observations suggests that the CF IA complex could be playing a general role in the termination of transcription in budding yeast. More direct evidence, however, was needed in support of a role for the CF IA complex in the termination of transcription on a genomewide scale and to firmly establish it as a general termination factor.The CF IA 3′ end processing complex of budding yeast is composed of four subunits: Rna14, Rna15, Pcf11 and Clp1 as shown in Fig. 9. In yeast, the 5′ end initiated antisense transcripts are rapidly degraded by the RNA surveillance machinery of the cell and are therefore referred to as ‘cryptic unstable transcripts’ (CUTs)10. In contrast, the 3′ end initiated antisense transcripts are stable and belong to the category of ‘stable unannotated transcripts’ (SUTs)8. At least 50% of SUTs in budding yeast are 3′ end initiated antisense transcripts. A number of genes in yeast and mammalian cells exhibit 3′ end initiated antisense transcription under repressed conditions. The GAL10 gene of budding yeast, for example, exhibits robust sense transcription in the presence of galactose12. Upon shifting of cells to glucose containing medium, however, sense transcription is almost completely inhibited, and the 3′ end initiated antisense transcription predominates. It has been proposed that when the 3′ end initiated antisense transcript reaches the promoter end of the gene, it may adversely affect initiation/reinitiation either through transcriptional interference or through histone modifications15. Whether 3′ end initiated antisense transcription is a general feature of termination defective mutants is not clear.A vast majority of RNAPII-transcribed genes in yeast and higher eukaryotes exhibit antisense transcription initiating from both their 5′ as well as the 3′ ends6. Furthermore, the Ssu72 subunit of the CPF complex, which is also linked to 3′ end processing and termination of transcription in budding yeast, has been localized to the promoter end of genes17. The presence of termination factors towards the 5′ end of genes is an evolutionarily conserved feature as a number of 3′ end processing/termination factors of higher eukaryotes also have been found crosslinked to the distal ends of genes18. The promoter occupancy of termination factors remained an enigma until recently when it was demonstrated that these factors provide directionality to transcription by limiting divergent antisense transcription initiating from the promoter region20. The nucleosome free region located at the 5′ end of a majority of protein coding genes in yeast and mammals contains two juxtaposed unidirectional promoters25. The transcription from these promoters initiates in both the downstream sense direction as well as the upstream antisense direction. The downstream sense transcription of the coding region produces mRNA, while the upstream antisense transcription produces upstream antisense RNA (uaRNA)19. Transcription in the upstream antisense direction is terminated within a few hundred bases from the transcription start site by the termination factors residing in the promoter region, while productive transcription in the sense direction is allowed to proceed until the polymerase reaches the 3′ end of the gene26. Thus, promoter linked termination factors provide directionality to the inherently bidirectional eukaryotic promoter region by limiting synthesis of uaRNA. It has been demonstrated that gene looping plays a crucial role in the termination of uaRNA synthesis in budding yeast10. We recently showed that the CF IA complex is involved in providing transcription directionality during transcription of selected yeast genes5. Whether the CF IA complex has a general role in conferring promoter directionality during RNAPII-mediated transcription, however, needed further investigation.Since the CF IA complex is involved in the cleavage-polyadenylation of mRNA and termination of transcription, it is expected to be present near the 3′ end of genes. It was, however, intriguing to find all four subunits of the CF IA complex occupying the 5′ end of genes as wellCHA1 gene in budding yeast5. Here we extend these studies to show that the CF IA complex has a global role in the poly(A)-dependent termination of transcription. Employing the GRO-Seq approach, we further showed that the CF IA complex also influence promoter directionality of subset of yeast genes. How the CF 1A complex affects promoter-associated transcription, however, remains to be elucidated.Using a temperature-sensitive mutant of Clp1, we recently demonstrated the role of the CF IA complex in termination of transcription as well as in the promoter directionality of the 28. In addition, the Rat1 complex is also required to accomplish the termination step. When termination is defective, RNAPII does not dissociate from the template beyond the poly(A) site, but reads through the poly(A) termination signal29. This transcription readthrough phenotype is characteristic of defective termination, and has been widely used to determine the involvement of a factor in the termination of transcription. A reliable approach used for detecting the terminator readthrough phenotype is the nuclear run-on (NRO) assay4. This assay measures the density of transcriptionally active polymerase over a specific genomic locus. The genomewide version of this assay, called ‘Global Run-On-Seq (GRO-Seq), overcomes the limitations in the NRO assay in genome coverage and the strand specificity21. In GRO-Seq, the newly synthesized transcripts incorporate BrUTP, which allow for the affinity purification of these nascent transcripts on an anti-BrUTP column. The resultant nascent RNAs are subjected to high throughput sequencing. The outcome of GRO-Seq is a snapshot of the position as well as the density of the actively engaged polymerase in a strand specific manner.A combination of biochemical and genetic approaches has identified two macromolecular complexes called CF I and CPF being essential for both 3′ end processing and poly(A)-dependent termination of transcription in budding yeast Fig. 2, 27, 28To assess the role of the CF IA subunit Clp1 in transcription on a genomewide scale, we used the GRO-Seq approach. Comparing the GRO-Seq maps of the temperature-sensitive mutant of Clp1 and the isogenic wild type strain at the permissive (25 °C) and non-permissive (37 °C) temperatures of the mutant can reveal if Clp1 is a universal termination factor like the general transcription factors, or its role in termination is restricted to a subset of genes. Accordingly, we performed GRO-Seq analysis in the Clp1 mutant and the isogenic wild type strains in the cells grown at 25 °C and 37 °C. The experiments with the Clp1 mutant were performed in triplicates named TS-1, TS-2 and TS-3. The number of transcriptionally active genes was determined by using an experimentally determined threshold of 5 reads per kilo base. We found that ~5500 of the 6693 annotated ORFs (~82%) in the Saccharomyces Genome Database (SGD) were expressed as tags above the background. The heatmap of pairwise Pearson Correlation Coefficients among all samples shows that all TS-37 samples  are clustered together, while TS-25 , WT-25 and WT-37 samples are clustered together  and GPI18 (YBR004C) genes, which exhibit the termination defect in the GRO-Seq analysis in clp1 mutant at 37 °C  in the mutant and wild type cells grown at 25 °C and 37 °C. RTI was calculated by dividing the number of reads beyond the 3′ end of a genes with the number of reads before the 3′ end within the coding region. A termination defect will result in more GRO-Seq reads downstream of the 3′ end of a genes and therefore a higher RTI value. Accordingly, RTI value increased by about 2-fold in the mutant at 37 °C compared to at 25 °C Fig. . No suchNext, we examined if Clp1 is required for poly(A)-dependent termination of transcription only, or for the poly(A)-independent termination as well. The transcription termination of the snoRNA genes occurs in a poly(A)-independent manner. Most of the genes coding for snoRNA are located in the crowded region of yeast genome. We, therefore, looked at the termination of three snoRNA genes, snR13, snR63 and snR68, which are separated from the neighboring genes by several hundred base pairs. No termination defect was observed for these three genes in the clp1 mutant at elevated temperature Fig. . These rA number of genes both in yeast and mammalian systems exhibit 3′ initiated antisense transcription upon defective termination. It has been shown that the 3′ initiated antisense transcription inhibits the recruitment of transcription machinery at the promoter region for initiation of transcription. We, therefore, examined the role of Clp1 in antisense transcription originating from the 3′ end of genes. We choose the same set of 1038 genes for this analysis whose 5′ and 3′ ends were at least 400 bp away from the neighboring gene. Alignment of GRO-Seq reads to the 3′ end of these genes revealed enhanced antisense transcription in the Clp1 mutant at 37 °C compared to at 25 °C Fig. . No 3′ eCHA1 by facilitating reinitiation and conferring promoter directionality5. We, therefore, examined if Clp1 is facilitating initiation/reinitiation of transcription on a genomewide scale. If Clp1 was facilitating initiation/reinitiation of transcription, we expected a decrease in the density of mRNA transcribing polymerase in the mutant at 37 °C. When we mapped GRO-Seq reads of the mutant and the wild type cells grown at 37 °C, to the annotated 5′ end of genes, there was no decrease in the GRO-Seq signal for sense transcribing polymerase in the mutant at elevated temperature , while remaining approximately 60% of the transcriptionally active genes analyzed in this study did not exhibit any change in promoter directionality at elevated temperature. The overall conclusion of these results is that Clp1 influences the promoter directionality of nearly 40% of transcriptionally active genes analyzed in our study. These include both highly expressed and lowly expressed genes.To compute the number of genes exhibiting a decrease in promoter directionality in terms of enhanced uaRNA transcription in clp1 mutant at elevated temperature, we calculated ratio of GRO-Seq reads in 100 bp promoter upstream region in the antisense direction for individual genes in the mutant at 37 °C and 25 °C. We computed the difference in CPM (Count Per Million) reads in the mutant at the permissive and non-permissive temperatures as described in ‘Methods’ section. Our results indicate that of around 700 transcriptionally active genes analyzed in this study, 330 genes exhibited the CPM difference greater than +1 , while only 13 genes had CPM difference less than −1 Fig. . These rin vitro conditions. In contrast, termination is the least understood step of the transcription cycle. The accessory factors required for termination are not thoroughly characterized. Consequently, termination of transcription under in vitro conditions using purified yeast factors has not been achieved so far in budding yeast. A thorough understanding of the process of termination by RNAPII necessitates identification of the general termination factors in line with the general transcription factors that are required for initiation of transcription. A number of approaches like PAR-CLIP, GRO-Seq, PRO-Seq, and Net-Seq can be used to determine the genomewide role of an identified factor in termination of transcription. All these approaches may give similar results, but there are subtle differences in the precise information they reveal. We used GRO-Seq approach because of its high resolution and sensitivity in detecting defective termination.Of the three steps of the RNAPII transcription cycle , initiation is the most well understood step. The general initiation factors are well characterized, and it is possible to perform initiation of transcription from a defined promoter using highly purified factors under 30. A similar genomewide analysis was performed for CstF64, which is the mammalian homologue of another CF 1A subunit Rna1519. This analysis identified CstF64 as a general termination factor in mammalian RNAPII transcription cycle. Thus, the role of CF 1A complex and its mammalian counterpart in termination of transcription has been conserved during evolution. Besides CF 1A complex, termination of transcription in yeast requires CPF complex. It was recently demonstrated using PAR-CLIP approach that the CPF subunit Ysh1 is required for global termination of transcription31. The termination defective phenotype of Ysh1, however, was quite different from that of Clp1. Unlike Clp1, depletion of Ysh1 did not lead to readthrough transcription, but resulted in the accumulation of polymerase beyond the poly(A) site. These results suggested a role for Ysh1 in dissociating polymerase from the template beyond the poly(A) site. In contrast, CF 1A complex could be facilitating slowing down of polymerase near the poly(A) site, which is an important step in termination of transcription by RNAPII.An important conclusion of the GRO-Seq analysis performed here is that Clp1 is required for termination of transcription of RNPII-transcribed genes in budding yeast. Our analysis further show that Clp1 is required for poly(A)-dependent termination of transcription of relatively highly expressed genes. Since there is no report of Clp1 acting alone or being a part of a complex other than CF IA, we extrapolate these results to propose that the whole CF IA complex, and not its individual components separately, is required for termination of transcription. Our results are corroborated by a recent finding that another CF 1 A subunit Pcf11 is required for termination of transcription on a genomewide scale32. The proximity of the promoter and terminator regions in a gene loop allows promoter-bound factors to influence termination of transcription36. The gene looping also invokes the possibility of termination factors affecting the promoter-associated transcription. Accordingly, it was demonstrated that Ssu72, which is a component of CPF termination complex, affects directionality of promoter-initiated transcription10. We also showed that Clp1 affects transcription directionality of CHA15. Clp1 is also required for gene looping of CHA15. The results of this genomewide analysis demonstrate that, apart from its role in termination, Clp1 also function in regulating promoter directionality of RNAPII-transcribed yeast genes. Clp1 suppresses the promoter-initiated upstream antisense transcription of 330 out of about 700 transcriptionally active genes used in this analysis. The promoter-initiated downstream antisense transcription either remained unaffected or exhibited a marginal increase for a majority of genes in the absence of Clp1 activity. Thus, the role of Clp1 in promoter directionality is similar to that of CPF subunit Ssu7210. CAF-1 complex, which is associated with chromatin assembly pathway, also affected promoter-initiated divergent non-coding transcription of yeast genes in a manner similar to Clp137. Both Ssu72 and CAF-1 complex suppress promoter-initiated uaRNA transcription. The transcription of promoter-associated mRNA, however, either remains unaffected or exhibited a small increase in the absence of Ssu72 or CAF-1 activities. The NET-Seq analysis performed in mammalian cells likewise revealed the role of CstF64 and Xrn2 termination factors in transcription directionality19.During transcription, the distal ends of a gene physically interact with each other forming a looped gene architectureOverall conclusion of these results is that the CF 1A complex plays a role in poly(A)-coupled termination of transcription, and in limiting promoter-associated divergent antisense transcription on a genomewide scale in budding yeast. Not all RNAPII-transcribed yeast genes, however, are dependent on CF 1A complex for either termination of their transcription or promoter directionality. This is expected as there is an alternate pathway for termination of transcription in budding yeast that requires Nrd1-Nab3-Sen1 complex. Nrd1-dependent termination has been shown to affect termination of non-polyadenylated mRNA as well as uaRNA belonging to the category of CUTs. Is it gene looping that governs if termination of transcription and promoter directionality will be accomplished by the CF 1 A complex or the Nrd1 complex remains to be elucidated?MATa his3Δ200 trp1Δ63 leu2Δ0 met15Δ0 ura3Δ0) and clp1-769-5 (MATa ura3Δ0 leu2Δ0 his3Δ1lys2Δ0can1Δ::LEU2-MFA1pr::His3 clp1ts::URA3).The yeast strains used in this study are BY4733  medium to an OD600 of 0.4. The 100 ml culture was transferred to two 50 ml sterile tubes, and centrifuged at 1860 × g for 5 minutes. Cell pellets were resuspended in 50 ml of 25 °C or 37 °C prewarmed YPD medium, and grown at the permissive (25 °C) or non-permissive temperature (37 °C) for 2 hours. This normally takes cells to an OD600~0.8. Equal number of cells were pelleted by centrifugation at 1860 × g for 5 minute at 4 °C. Cell pellets were resuspended in 10 ml of ice-cold TMN buffer , incubated in ice for 10 minutes, centrifuged at 1860 × g for 5 minutes, and then resuspended in 940 μl of DEPC (Diethylpyrocarbonate)-treated ice-cold water. Cells were permeabilized by incubating with 60 μl 10% sarkosyl at 4 °C for 25 minutes with gentle shaking. Permeabilized cells were spun down at 1200 × g for 6 minutes at 4 °C. The pellets were stored at 4 °C till all samples were ready for the next step.The et al., (1997). Cell pellets from step I above were resuspended in 150 μl of transcription run-on reaction buffer  containing 5 μl of freshly added RNase Inhibitor (40 units/μl) and incubated at 30 °C for 5 minutes. The reaction was stopped by adding 500 μl of ice-cold Trizol reagent. About 250 μl of acid-washed glass beads were added to each tube. Cells were immediately lysed by vigorous shaking at 4 °C for 20 minutes. The lysate was transferred to a new 1.5 ml microfuge tube containing 500 μl of Trizol and 200 μl of chloroform. The tubes were shaken vigorously and centrifuged at 14000 rpm at 25 °C for 20 minutes. The supernatant was extracted with phenol-chloroform three times. Total RNA was precipitated by adding 3 volumes of ice-cold 100% ethanol, 2 μl of glycogen (20 mg/ml) and NaCl to a final concentration of 0.3 M followed by overnight incubation at −20 °C. The precipitated RNA was recovered by centrifugation at 16000 × g for 30 minutes at 4 °C. The RNA pellet was washed once with ice-cold 75% ethanol, air dried and resuspended in 55 μl of DEPC-treated water.Transcription run-on reaction was performed by the modification of methods described in Birse The extracted RNA was subjected to partial hydrolysis by incubating with 500 mM NaOH on ice for 20 minutes. The NaOH was neutralized by adding 30 μl of 1 M Tris-HCl (pH 6.6). Partially hydrolyzed RNA was purified using RNeasy kit (Qiagen) to remove any unincorporated nucleotides and to exchange buffer. RNA was eluted from the RNeasy column twice with 50 μl of DEPC treated water.About 25 μl of anti-BrdU-antibody-conjugated agarose beads were washed three times with 500 μl of binding buffer . Beads were blocked by gently shaking with 500 μl of blocking buffer (485 μl binding buffer containing 5 μl of 10% polyvinylpyrrolidone and 10 μl of Ultrapure BSA) for two hours at 4 °C. The blocked beads were washed twice with 500 μl of binding buffer and then resuspended in 400 μl of the same buffer. The RNA from step II was incubated at 65 °C for 5 minutes to get rid of any secondary structure and immediately placed on ice for 2 minutes. The RNA was then bound to the processed anti-BrdU beads with gentle shaking for one hour at 4 °C. Beads were then washed sequentially with 500 μl of binding buffer, 500 μl of low salt buffer  and 500 μl of high salt buffer  once each, followed by two washes of 500 μl of TET buffer . All washes were performed on nutator for three minutes each. All centrifugation steps between the washes were at 194 × g for 1 minute each, with incubation of tubes on ice for 30 seconds before removing wash buffer. The bound nascent RNA was eluted two times with 125 μl and one time with 250 μl of elution buffer  for a total elution time of 10 minutes in 42 °C water bath. The eluted RNA was precipitated with NaCl as described previously.RNA pellet was washed with ice-cold 75% ethanol, air dried and resuspended in 12 μl of DEPC-treated water and quantified using a Nanodrop spectrophotometer.TM complete Kit (Epicenter) following manufacturer’s instructions. DNA Sequencing was performed at the DNA Core Facility of Cornell University.The nascent, purified RNA samples were first depleted of rRNA. The depletion of rRNA was performed by the Ribo-Zero™ Magnetic Kit (Epicenter) following manufacturer’s recommendations. GRO-Seq libraries were constructed using the ScriptSeq38 for quality control of raw fastq files to trim adapters and remove low quality reads. All high quality fastq files were mapped to Saccharomyces genome  using Tophat239  and HTSeq.40 (version 0.6) for calculating read counts. Subsequently DESeq. 2 was performed for read count normalization. Then reads per kilobase of transcript per million (RPKM) mapped reads > = 5 RPKM was done for determining number of expressed genes41.We used CutadaptPheatmap package is applied to generate pairwise Pearson correlation coefficients analysis on normalized count files, and ggplot2 package applied for creating pairwise scatter plot among all replicates.To calculate ‘Read Through Index’ (RTI) score and expression difference of samples regards to their coding strand, again all high quality fastq files were mapped to three modified Gene Transfer format (GTF) files TSS, TTS and gene body regions of Saccer3 genome by Tophat2. In addition HTSeq and DESeq. 2 applied to obtain normalized read count. The output is used to calculate RTI score for each gene as shown in Fig. All scripts for calculating RTI were performed using R and ggplot2 package employed to generate density plot s and scatter plots.To determine the number of genes showing loss of promoter directionality due to increased uaRNA transcription in the mutant at 37 °C, we computed the count of reads in each sample in the 100 bp non-coding window upstream to the TSS. We performed cross-sample normalization by dividing the RAW count values to the total number of aligned reads (in millions) per sample to compute Count Per Million (CPM) reads. Then we computed the Log2 fold-change of mean CPM values between TS-37 vs. TS-25 samples and the sum of CPM values for TS-37 samples minus TS-25 samples. The CPM difference greater than +1 indicates more than 2-fold higher expression in TS-37 samples, while a CPM difference less than −1 shows more than 2-fold higher expression in TS-25 sample. The mean CPM difference value for all genes is +1.136, and the median value is +0.545.The data present in this work was deposited in NCBI’s Gene Expression Omnibus (GEO) database under the accession number GSE71624.Supplemental Information


Essays Biochem. (2016) volume 60, issue 3, pages 255–273;https://doi.org/10.1042/EBC20160016The penultimate sentence of article was factually incorrect as published; the corrected sentence is presented here (corrections underlined):2 into malate during the night via PEP carboxylase, store it within the vacuole of the plant cell at night and in the daytime the malate is transported to chloroplasts to be fixed via normal C3 photosynthesis.”“The Crassulaceae fix CO