F-actin Turnover Research Articles

RhoA regulates oligodendrocyte differentiation and myelination by orchestrating cortical and membrane tension.

Timely differentiation and myelin formation by oligodendrocytes are essential for the physiological functioning of the central nervous system (CNS). While the Rho GTPase RhoA has been hinted as a negative regulator of myelin sheath formation, the precise in vivo mechanisms have remained elusive. Here we show that RhoA controls the timing and progression of myelination by oligodendrocytes through a fine-tuned balance between cortical tension, membrane tension and cell shape. Using a conditional mouse model, we observe that Rhoa ablation results in the acceleration of myelination driven by hastened differentiation and facilitated through membrane expansion induced by changes in MLCII activity and in F-actin redistribution and turnover within the cell. These findings reveal RhoA as a central molecular integrator of alterations in actin cytoskeleton, actomyosin contractility and membrane tension underlying precise morphogenesis of oligodendrocytes and normal myelination of the CNS.

Emergent actin flows explain distinct modes of gliding motility

AbstractDuring host infection, Toxoplasma gondii and related unicellular parasites move using gliding, which differs fundamentally from other known mechanisms of eukaryotic cell motility. Gliding is thought to be powered by a thin layer of flowing filamentous (F)-actin sandwiched between the plasma membrane and a myosin-covered inner membrane complex. How this surface actin layer drives the various gliding modes observed in experiments—helical, circular, twirling and patch, pendulum or rolling—is unclear. Here we suggest that F-actin flows arise through self-organization and develop a continuum model of emergent F-actin flow within the confines provided by Toxoplasma geometry. In the presence of F-actin turnover, our model predicts the emergence of a steady-state mode in which actin transport is largely directed rearward. Removing F-actin turnover leads to actin patches that recirculate up and down the cell, which we observe experimentally for drug-stabilized actin bundles in live Toxoplasma gondii parasites. These distinct self-organized actin states can account for observed gliding modes, illustrating how different forms of gliding motility can emerge as an intrinsic consequence of the self-organizing properties of F-actin flow in a confined geometry.

Cytosolic concentrations of actin binding proteins and the implications for in vivo F-actin turnover.

Understanding how numerous actin-binding proteins (ABPs) work in concert to control the assembly, organization, and turnover of the actin cytoskeleton requires quantitative information about the levels of each component. Here, we measured the cellular concentrations of actin and the majority of the conserved ABPs in Saccharomyces cerevisiae, as well as the free (cytosolic) fractions of each ABP. The cellular concentration of actin is estimated to be 13.2 µM, with approximately two-thirds in the F-actin form and one-third in the G-actin form. Cellular concentrations of ABPs range from 12.4 to 0.85 µM (Tpm1> Pfy1> Cof1> Abp1> Srv2> Abp140> Tpm2> Aip1> Cap1/2> Crn1> Sac6> Twf1> Arp2/3> Scp1). The cytosolic fractions of all ABPs are unexpectedly high (0.6-0.9) and remain so throughout the cell cycle. Based on these numbers, we speculate that F-actin binding sites are limited in vivo, which leads to high cytosolic levels of ABPs, and in turn helps drive the rapid assembly and turnover of cellular F-actin structures.

Actin depolymerizing factor ADF7 inhibits actin bundling protein VILLIN1 to regulate root hair formation in response to osmotic stress in Arabidopsis.

Actin cytoskeleton is essential for root hair formation. However, the underlying molecular mechanisms of actin dynamics in root hair formation in response to abiotic stress are largely undiscovered. Here, genetic analysis showed that actin-depolymerizing protein ADF7 and actin-bundling protein VILLIN1 (VLN1) were positively and negatively involved in root hair formation of Arabidopsis respectively. Moreover, RT-qPCR, GUS staining, western blotting, and genetic analysis revealed that ADF7 played an important role in inhibiting the expression and function of VLN1 during root hair formation. Filament actin (F-actin) dynamics observation and actin pharmacological experiments indicated that ADF7-inhibited-VLN1 pathway led to the decline of F-actin bundling and thick bundle formation, as well as the increase of F-actin depolymerization and turnover to promote root hair formation. Furthermore, the F-actin dynamics mediated by ADF7-inhibited-VLN1 pathway was associated with the reactive oxygen species (ROS) accumulation in root hair formation. Finally, ADF7-inhibited-VLN1 pathway was critical for osmotic stress-induced root hair formation. Our work demonstrates that ADF7 inhibits VLN1 to regulate F-actin dynamics in root hair formation in response to osmotic stress, providing the novel evidence on the F-actin dynamics and their molecular mechanisms in root hair formation and in abiotic stress.

Modeling cell protrusion predicts how myosin II and actin turnover affect adhesion-based signaling

Orchestration of cell migration is essential for development, tissue regeneration, and the immune response. This dynamic process integrates adhesion, signaling, and cytoskeletal subprocesses across spatial and temporal scales. In mesenchymal cells, adhesion complexes bound to extracellular matrix mediate both biochemical signal transduction and physical interaction with the F-actin cytoskeleton. Here, we present a mathematical model that offers insight into both aspects, considering spatiotemporal dynamics of nascent adhesions, active signaling molecules, mechanical clutching, actin treadmilling, and nonmuscle myosin II contractility. At the core of the model is a positive feedback loop, whereby adhesion-based signaling promotes generation of barbed ends at, and protrusion of, the cell’s leading edge, which in turn promotes formation and stabilization of nascent adhesions. The model predicts a switch-like transition and optimality of membrane protrusion, determined by the balance of actin polymerization and retrograde flow, with respect to extracellular matrix density. The model, together with new experimental measurements, explains how protrusion can be modulated by mechanical effects (nonmuscle myosin II contractility and adhesive bond stiffness) and F-actin turnover.

MRCKα Is Dispensable for Breast Cancer Development in the MMTV-PyMT Model.

MRCKα is a ubiquitously expressed serine/threonine kinase involved in cell contraction and F-actin turnover, which is highly amplified in human breast cancer and part of a gene expression signature for bad prognosis. Nothing is known about the in vivo function of MRCKα. To explore MRCKα function in development and in breast cancer, we generated mice lacking a functional MRCKα gene. Mice were born close to the Mendelian ratio and showed no obvious phenotype including a normal mammary gland formation. Assessing breast cancer development using the transgenic MMTV-PyMT mouse model, loss of MRCKα did not affect tumor onset, tumor growth and metastasis formation. Deleting MRCKα and its related family member MRCKβ in two triple-negative breast cancer cell lines resulted in reduced invasion of MDA-MB-231 cells, but did not affect migration of 4T1 cells. Further genomic analysis of human breast cancers revealed that MRCKα is frequently co-amplified with the oncogenes ARID4B and AKT3 which might contribute to the prognostic value of MRCKα expression. Collectively, these data suggest that MRCKα might be a prognostic marker for breast cancer, but probably of limited functional importance.

\u03b1-Linolenic acid induces clearance of Tau seeds via Actin-remodeling in Microglia

Alzheimer’s disease (AD) is known by characteristic features, extracellular burden of amyloid-β and intracellular neuronal Tau. Microglia, the innate immune cell of the brain has the ability to clear the burden of accumulated proteins via phagocytosis. But the excessive proinflammatory cytokine production, altered cellular signaling and actin remodeling hampers the process of migration and phagocytosis by microglia. Actin remodeling is necessary to initiate the chemotactic migration of microglia towards the target and engulf it. The formation of lamellipodia, filopodia, membrane ruffling and rapid turnover of F-actin is necessary to sense the extracellular target by the cells. Omega-3 fatty acids, are known to impose anti-inflammatory phenotype of microglia by enhancing its ability for migration and phagocytosis. But the role of omega-3 fatty acids in cellular actin remodeling, which is the basis of cellular functions such as migration and phagocytosis, is not well understood. Here, we have focused on the effect of dietary supplement of α-linolenic acid (ALA) on extracellular Tau internalization and assisted actin polymerization for the process. ALA is found to induce membrane ruffling and phagocytic cup formation along with cytoskeletal rearrangement. ALA also enhances the localization of Arp2/3 complex at the leading edge and its colocalization with F-actin to induce the actin polymerization. The excessive actin polymerization might help the cell to protrude forward and perform its migration. The results suggest that dietary supplement of ALA could play a neuroprotective role and slow down the AD pathology.

Twinfilin uncaps filament barbed ends to promote turnover of lamellipodial actin networks.

Coordinated polymerization of actin filaments provides force for cell migration, morphogenesis and endocytosis. Capping protein (CP) is a central regulator of actin dynamics in all eukaryotes. It binds to actin filament (F-actin) barbed ends with high affinity and slow dissociation kinetics to prevent filament polymerization and depolymerization. However, in cells, CP displays remarkably rapid dynamics within F-actin networks, but the underlying mechanism remains unclear. Here, we report that the conserved cytoskeletal regulator twinfilin is responsible for CP's rapid dynamics and specific localization in cells. Depletion of twinfilin led to stable association between CP and cellular F-actin arrays, as well as to its retrograde movement throughout leading-edge lamellipodia. These were accompanied by diminished F-actin turnover rates. In vitro single-filament imaging approaches revealed that twinfilin directly promotes dissociation of CP from filament barbed ends, while enabling subsequent filament depolymerization. These results uncover a bipartite mechanism that controls how actin cytoskeleton-mediated forces are generated in cells.

Sensing Actin Dynamics through Adherens Junctions

We study punctate adherens junctions (pAJs) to determine how short-lived cadherin clusters and relatively stable actin bundles interact despite differences in dynamics. We show that pAJ-linked bundles consist of two distinct regions-the bundle stalk (AJ-BS) and a tip (AJ-BT) positioned between cadherin clusters and the stalk. The tip differs from the stalk in a number of ways: it is devoid of the actin-bundling protein calponin, and exhibits a much faster F-actin turnover rate. While F-actin in the stalk displays centripetal movement, the F-actin in the tip is immobile. The F-actin turnover in both the tip and stalk is dependent on cadherin cluster stability, which in turn is regulated by F-actin. The close bidirectional coupling between the stability of cadherin and associated F-actin shows how pAJs, and perhaps other AJs, allow cells to sense and coordinate the dynamics of the actin cytoskeleton in neighboring cells-a mechanism we term "dynasensing."

Arp2/3 Is Required for Axonal Arbor Terminal Retraction in Cerebellar Granule Neurons

Although selective pruning of subsets of branches is a key step for axonal remodeling, the underlying mechanisms have not fully elucidated. We have reported that the retraction rate of axonal arbor is higher in the terminals, where less kinesin is sorted, and inhibiting actin turnover by treatment with latrunculin A prevented retraction in such terminals. In the present study, we investigated the effects of pharmacological inhibitors for the regulators of actin polymerization/destabilization on the elongation and retraction of arbor terminals in cerebellar granule neurons. Inhibition of an actin nucleator Arp2/3 with CK-666 or CK-869 inhibited the retraction of arbor terminals. However, inhibitors for PAK/LIMK, upstream signaling of ADF/cofilin, reduced the elongation rate but not the retraction rate. A photoconversion analysis revealed that turnover of F-actin at the tip of the axonal terminal is faster in the presence of the Arp2/3 inhibitor compared with the control and neurons treated with the LIMK inhibitor. Our results indicate the important role of Arp2/3 for the retraction of axonal arbor terminals in cerebellar granule neurons.

Dynamic motions of molecular motors in the actin cytoskeleton.

During intracellular transport, cellular cargos, such as organelles, vesicles, and proteins, are transported within cells. Intracellular transport plays an important role in diverse cellular functions. Molecular motors walking on the cytoskeleton facilitate active intracellular transport, which is more efficient than diffusion-based passive transport. Active transport driven by kinesin and dynein walking on microtubules has been studied well during recent decades. However, mechanisms of active transport occurring in disorganized actin networks via myosin motors remain elusive. To provide physiologically relevant insights, we probed motions of myosin motors in actin networks under various conditions using our well-established computational model that rigorously accounts for the mechanical and dynamical behaviors of the actin cytoskeleton. We demonstrated that myosin motions can be confined due to three different reasons in the absence of F-actin turnover. We verified mechanisms of motor stalling using in vitro reconstituted actomyosin networks. We also found that with F-actin turnover, motors consistently move for a long time without significant confinement. Our study sheds light on the importance of F-actin turnover for effective active transport in the actin cytoskeleton.

Myosin XVI Regulates Actin Cytoskeleton Dynamics in Dendritic Spines of Purkinje Cells and Affects Presynaptic Organization.

The actin cytoskeleton is crucial for function and morphology of neuronal synapses. Moreover, altered regulation of the neuronal actin cytoskeleton has been implicated in neuropsychiatric diseases such as autism spectrum disorder (ASD). Myosin XVI is a neuronally expressed unconventional myosin known to bind the WAVE regulatory complex (WRC), a regulator of filamentous actin (F-actin) polymerization. Notably, the gene encoding the myosin’s heavy chain (MYO16) shows genetic association with neuropsychiatric disorders including ASD. Here, we investigated whether myosin XVI plays a role for actin cytoskeleton regulation in the dendritic spines of cerebellar Purkinje cells (PCs), a neuronal cell type crucial for motor learning, social cognition and vocalization. We provide evidence that both myosin XVI and the WRC component WAVE1 localize to PC spines. Fluorescence recovery after photobleaching (FRAP) analysis of GFP-actin in cultured PCs shows that Myo16 knockout as well as PC-specific Myo16 knockdown, lead to faster F-actin turnover in the dendritic spines of PCs. We also detect accelerated F-actin turnover upon interference with the WRC, and upon inhibition of Arp2/3 that drives formation of branched F-actin downstream of the WRC. In contrast, inhibition of formins that are responsible for polymerization of linear actin filaments does not cause faster F-actin turnover. Together, our data establish myosin XVI as a regulator of the postsynaptic actin cytoskeleton and suggest that it is an upstream activator of the WRC-Arp2/3 pathway in PC spines. Furthermore, ultra-structural and electrophysiological analyses of Myo16 knockout cerebellum reveals the presence of reduced numbers of synaptic vesicles at presynaptic terminals in the absence of the myosin. Therefore, we here define myosin XVI as an F-actin regulator important for presynaptic organization in the cerebellum.

Quantitative high-precision imaging of myosin-dependent filamentous actin dynamics.

Over recent decades, considerable effort has been made to understand how mechanical stress applied to the actin network alters actin assembly and disassembly dynamics. However, there are conflicting reports concerning the issue both in vitro and in cells. In this review, we discuss concerns regarding previous quantitative live-cell experiments that have attempted to evaluate myosin regulation of filamentous actin (F-actin) turnover. In particular, we highlight an error-generating mechanism in quantitative live-cell imaging, namely convection-induced misdistribution of actin-binding probes. Direct observation of actin turnover at the single-molecule level using our improved electroporation-based Single-Molecule Speckle (eSiMS) microscopy technique overcomes these concerns. We introduce our recent single-molecule analysis that unambiguously demonstrates myosin-dependent regulation of F-actin stability in live cells. We also discuss the possible application of eSiMS microscopy in the analysis of actin remodeling in striated muscle cells.

Cofilin drives rapid turnover and fluidization of entangled F-actin

The shape of most animal cells is controlled by the actin cortex, a thin network of dynamic actin filaments (F-actin) situated just beneath the plasma membrane. The cortex is held far from equilibrium by both active stresses and polymer turnover: Molecular motors drive deformations required for cell morphogenesis, while actin-filament disassembly dynamics relax stress and facilitate cortical remodeling. While many aspects of actin-cortex mechanics are well characterized, a mechanistic understanding of how nonequilibrium actin turnover contributes to stress relaxation is still lacking. To address this, we developed a reconstituted in vitro system of entangled F-actin, wherein the steady-state length and turnover rate of F-actin are controlled by the actin regulatory proteins cofilin, profilin, and formin, which sever, recycle, and assemble filaments, respectively. Cofilin-mediated severing accelerates the turnover and spatial reorganization of F-actin, without significant changes to filament length. We demonstrate that cofilin-mediated severing is a single-timescale mode of stress relaxation that tunes the low-frequency viscosity over two orders of magnitude. These findings serve as the foundation for understanding the mechanics of more physiological F-actin networks with turnover and inform an updated microscopic model of single-filament turnover. They also demonstrate that polymer activity, in the form of ATP hydrolysis on F-actin coupled to nucleotide-dependent cofilin binding, is sufficient to generate a form of active matter wherein asymmetric filament disassembly preserves filament number despite sustained severing.

Mechanisms for Dendritic Actin Network Formation, Distributed Turnover, and Structural Remodeling

The dendritic network of actin filaments provides the force for lamellipodial protrusions, driven by actin filament polymerization and branch generation by the Arp2/3 complex. Electron microscopy experiments of lamellipodia revealed that the network structure of filaments varies with distance to the leading edge. Near the leading edge there is a dense brushwork composed of short filaments. Filaments are longer and appear more linear near the center and rear of keratocyte lamellipodia. Prior modeling of FRAP and single molecule imaging experiments suggested the existence of a diffuse actin oligomer cytoplasmic pool and distributed turnover of F-actin through the lamellipodium. The precise mechanisms behind network remodeling and the role of the oligomer pool have yet to be determined. To answer this question, we created a three-dimensional stochastic model at the filament level that includes rate constants for known mechanisms of polymerization, depolymerization, branching, capping, uncapping, severing and debranching. The model reproduces the +/− 35o orientation pattern when branching occurs within 10o of the lamellipodial plane as well as the density of branches, ends and length distribution near the leading edge for both fibroblast and keratocyte lamellipodia (that differ in the magnitude of retrograde flow and polymerization rates). We show that the simplest implementations of severing, debranching, uncapping, and depolymerization from either pointed or barbed ends does not provide mechanisms for network structural remodeling at the level observed in electron micrographs. We examine and test several mechanisms that include severing and oligomer annealing that may provide an explanation.

F-actin homeostasis through transcriptional regulation and proteasome-mediated proteolysis

Many organisms possess multiple and often divergent actins whose regulation and roles are not understood in detail. For example, Chlamydomonas reinhardtii has both a conventional actin (IDA5) and a highly divergent one (NAP1); only IDA5 is expressed in normal proliferating cells. We showed previously that the drug latrunculin B (LatB) causes loss of filamentous (F-) IDA5 and strong up-regulation of NAP1, which then provides essential actin function(s) by forming LatB-resistant F-NAP1. RNA-sequencing analyses now show that this up-regulation of NAP1 reflects a broad transcriptional response, much of which depends on three proteins (LAT1, LAT2, and LAT3) identified previously as essential for NAP1 transcription. Many of the LAT-regulated genes contain a putative cis-acting regulatory site, the "LRE motif." The LatB transcriptional program appears to be activated by loss of F-IDA5 and deactivated by formation of F-NAP1, thus forming an F-actin-dependent negative-feedback loop. Multiple genes encoding proteins of the ubiquitin-proteasome system are among those induced by LatB, resulting in rapid degradation of IDA5 (but not NAP1). Our results suggest that IDA5 degradation is functionally important because nonpolymerizable LatB-bound IDA5 interferes with the formation of F-NAP1. The genes for the actin-interacting proteins cofilin and profilin are also induced. Cofilin induction may further the clearance of IDA5 by promoting the scission of F-IDA5, whereas profilin appears to function in protecting monomeric IDA5 from degradation. This multifaceted regulatory system allows rapid and quantitative turnover of F-actin in response to cytoskeletal perturbations and probably also maintains F-actin homeostasis under normal growth conditions.

Actin activates Pseudomonas aeruginosa ExoY nucleotidyl cyclase toxin and ExoY-like effector domains from MARTX toxins.

The nucleotidyl cyclase toxin ExoY is one of the virulence factors injected by the Pseudomonas aeruginosa type III secretion system into host cells. Inside cells, it is activated by an unknown eukaryotic cofactor to synthesize various cyclic nucleotide monophosphates. ExoY-like adenylate cyclases are also found in Multifunctional-Autoprocessing Repeats-in-ToXin (MARTX) toxins produced by various Gram-negative pathogens. Here we demonstrate that filamentous actin (F-actin) is the hitherto unknown cofactor of ExoY. Association with F-actin stimulates ExoY activity more than 10,000 fold in vitro and results in stabilization of actin filaments. ExoY is recruited to actin filaments in transfected cells and alters F-actin turnover. Actin also activates an ExoY-like adenylate cyclase MARTX effector domain from Vibrio nigripulchritudo. Finally, using a yeast genetic screen, we identify actin mutants that no longer activate ExoY. Our results thus reveal a new sub-group within the class II adenylyl cyclase family, namely actin-activated nucleotidyl cyclase (AA-NC) toxins.

Leishmaniainfection inhibits macrophage motility by altering F-actin dynamics and the expression of adhesion complex proteins

Leishmania is an intracellular protozoan parasite that causes a broad spectrum of clinical manifestations, ranging from self-healing skin lesions to fatal visceralizing disease. As the host cells of choice for all species of Leishmania, macrophages are critical for the establishment of infections. How macrophages contribute to parasite homing to specific tissues and how parasites modulate macrophage function are still poorly understood. In this study, we show that Leishmania amazonensis infection inhibits macrophage roaming motility. The reduction in macrophage speed is not dependent on particle load or on factors released by infected macrophages. L.amazonensis-infected macrophages also show reduced directional migration in response to the chemokine MCP-1. We found that infected macrophages have lower levels of total paxillin, phosphorylated paxillin, and phosphorylated focal adhesion kinase when compared to noninfected macrophages, indicating abnormalities in the formation of signaling adhesion complexes that regulate motility. Analysis of the dynamics of actin polymerization at peripheral sites also revealed a markedly enhanced F-actin turnover frequency in L.amazonensis-infected macrophages. Thus, Leishmania infection inhibits macrophage motility by altering actin dynamics and impairing the expression of proteins that function in plasma membrane-extracellular matrix interactions.

The F-actin modulator SWAP-70 controls podosome patterning in osteoclasts

Osteoclasts are bone resorbing cells acting as key mediators of bone disorders. Upon adhesion to bone, osteoclasts polarize and reorganize their cytoskeleton to generate a ring-like F-actin-rich structure, the sealing zone, wherein the osteoclast's resorptive organelle, the ruffled border, is formed. The dynamic self-organization of actin-rich adhesive structures, the podosomes, from clusters to belts is crucial for osteoclast-mediated bone degradation. Mice lacking the protein SWAP-70 display an osteopetrotic phenotype due to defective bone resorption caused by impaired actin ring formation in Swap-70−/− osteoclasts. To further elucidate the mechanisms underlying this defect, we investigated the specific function of SWAP-70 in the organization and dynamics of podosomes. These detailed studies show that the transition from podosome clusters to rings is impaired in Swap-70−/− osteoclasts. Live cell imaging of dynamic F-actin turnover and SWAP-70 localization during podosome patterning indicate that SWAP-70 is dispensable for cluster formation but plays a key role in F-actin ring generation. Our data provide insights in the role of SWAP-70's F-actin binding domain and pleckstrin homology (PH) domain in the proper localization of SWAP-70 and formation of a peripheral podosome belt, respectively. Ex vivo bone analyses revealed that SWAP-70-deficient osteoclasts exhibit defective ruffled border formation and V-ATPase expression. Our findings suggest an important role of membrane binding of SWAP-70 for the regulation of actin dynamics, which is essential for podosome patterning, and thus for the resorptive activity of osteoclasts.

Cold-Stored Platelets in PAS Exhibit Superior Hemostatic Potential

Background: Platelet transfusion is vital for hemostasis in patients with severe bleeding. Currently, platelets are stored at room temperature (RT) for 5 days with gentle agitation. However, this method results in a platelet storage lesion characterized by loss of platelet function and elevated risk of bacterial contamination. It has been shown that refrigeration of platelets at 4o C mitigates adverse effects and other logistical difficulties. Recently, FDA approved the use of cold-stored apheresis platelets for the resuscitation of actively bleeding patients for up to 3 days (5. 21 CFR 606.65(e) & 610.53(c)). We hypothesized that the refrigeration of platelets stored in 65% platelet additive solution (PAS), would better preserve the platelet contractile apparatus and overall hemostatic function. We measured clot stiffness, strength, crosslinking density and platelet contractile forces (PCF) using cone and plate rheometry; and quantified the microstructural architecture of the clots. To corroborate the rheological and morphological measurements with the clotting mechanism, we assayed thrombin generation, FXIII activity, clot retraction, and cytoskeletal rearrangement.Methods: Apheresis platelet (AP) concentrates collected from 4 healthy donors were isolated and stored in 65% Platelet additive solution (PAS) for 5 days at RT (22-24°C) or 4C (4°C). Clotting function was assayed after re-suspension in fresh frozen plasma (FFP). After initiating clotting with 20mM Ca2+, 100µL of the PRP sample was placed on the rheometer plate and clot stiffness was measured for 30 mins at 37o C at a small strain of 1Hz by tracking the evolving storage modulus (G'). Normal force measurements were made to quantify the force generated in the samples as a result of platelet contractile forces. Subsequently, a progressively increasing strain of 1 to 250% was applied until the clot was irreversibly deformed to estimate the yield point (clot strength). Scanning Electron Microscopy was performed on the clots from the rheological analysis to study the clot microstructure. Thrombin generation and FXIII activity were determined using Calibrated Automated Thrombogram and activity assays, respectively. Further, the cytoskeletal rearrangement was studied by activating the washed platelets with thrombin/ TRAP for 5, 10 and 15 seconds and estimating the G-actin to F-actin turnover by Western Blots. The results were analyzed using one-way ANOVA and a post hoc Tukey test for multiple comparisons. The groups were considered statistically significant for p values less than 0.05.Results: The rheological studies on clotting indicate that cold stored platelets form clots that have comparable properties to fresh platelets. RT-stored platelets formed weaker clots (Fig. 1A, B). These findings supported by the evidence of increased FXIII cross-linking activity (Fig.1C). Further, the normal force measurements indicate reduced force generation in RT-stored platelets which imply decreased platelet contractility (Fig. 1D). We also observed differences in geometry and orientation of individual fibrin fibers between fresh and stored platelets. Our preliminary results suggest that thrombin generation potential, crosslinking density, and platelet contractility may contribute to the mechanical strength and morphological differences between fresh and platelets stored at cold and room temperature.Conclusion: Cold-stored platelets form structurally and mechanically stable clots compared to 5 day stored RT platelets as demonstrated by the rheological and microstructural studies. Together, these data suggest that 4C platelets are hemostatically more active than the current standard-of-care and may offer a better solution to patients for resuscitation of bleeding trauma patients.Figure 1 Mechanical testing of evolving clot by rheometry. (A) RT-stored platelets form clots with decreased clot stiffness (G') compared to fresh (F) and cold-stored (4C) platelets (B) Clot strength was defined as the yield point where the clot deformed irreversibly. Clots formed from RT-stored platelets offer less resistance to strain; (C) Relative Cross Linking Density was estimated as the slope of stress-strain curve obtained from amplitude sweep. RT-stored platelet clots show less crosslinking; (D) less contractile force generation in RT platelets. * p <0.05, n=4. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.