Myeloid leukemias, diseases marked by aggressiveness and poor outcomes, are frequently triggered by oncogenic translocations. In the case of chronic myelogenous leukemia (CML) the BCR-ABL fusion initiates chronic phase disease with second hits allowing progression to blast crisis. Although Gleevec has been transformative for CML, blast crisis CML remains relatively drug resistant. Here we show that MSI2-HOXA9, a translocation with an unknown role in cancer, can serve as a second hit in driving bcCML. Compared to BCR-ABL, BCR-ABL/MSI2-HOXA9 led to a more aggressive diseasein vivowith decreased latency, increased lethality and a differentiation blockade that is a hallmark of blast crisis. Domain mapping revealed that the MSI2 RNA binding domain RRM1 had a preferential impact on growth and lethality of bcCML relative to RRM2 or the HOXA9 domain. Mechanistically, MSI2-HOXA9 triggered global downstream changes with a preferential upregulation of mitochondrial components. Consistent with this, BCR-ABL/MSI2-HOXA9 cells exhibited a significant increase in mitochondrial respiration. These data suggest that MSI2-HOXA9 acts, at least in part, by increasing expression of the mitochondrial polymerase Polrmt and augmenting mitochondrial function and basal respiration in blast crisis. Collectively, our findings demonstrate for the first time that translocations involving the stem and developmental signal MSI2 can be oncogenic, and suggest that MSI, which we found to be a frequent partner for an array of translocations, could also be a driver mutation across solid cancers.

Aggressive myeloid leukemias such as blast crisis chronic myeloid leukemia and acute myeloid leukemia remain highly lethal. Here we report a genome-wide in vivo CRISPR screen to identify new dependencies in this disease. Among these, RNA-binding proteins (RBPs) in general, and the double-stranded RBP Staufen2 (Stau2) in particular, emerged as critical regulators of myeloid leukemia. In a newly developed knockout mouse, loss of Stau2 led to a profound decrease in leukemia growth and improved survival in mouse models of the disease. Further, Stau2 was required for growth of primary human blast crisis chronic myeloid leukemia and acute myeloid leukemia. Finally, integrated analysis of CRISPR, eCLIP and RNA-sequencing identified Stau2 as a regulator of chromatin-binding factors, driving global alterations in histone methylation. Collectively, these data show that in vivo CRISPR screening is an effective tool for defining new regulators of myeloid leukemia progression and identify the double-stranded RBP Stau2 as a critical dependency of myeloid malignancies.

Intratumoral heterogeneity is a common feature of many myeloid leukemias and a significant reason for treatment failure and relapse. Thus, identifying the cells responsible for residual disease and leukemia re-growth is critical to better understanding how they are regulated. Here, we show that a knock-in reporter mouse for the stem cell gene Musashi 2 (Msi2) allows identification of leukemia stem cells in aggressive myeloid malignancies, and provides a strategy for defining their core dependencies. Specifically, we carry out a high throughput screen using Msi2-reporter blast crisis chronic myeloid leukemia (bcCML) and identify several adhesion molecules that are preferentially expressed in therapy resistant bcCML cells and play a key role in bcCML. In particular, we focus on syndecan-1, whose deletion triggers defects in bcCML growth and propagation and markedly improves survival of transplanted mice. Further, live imaging reveals that the spatiotemporal dynamics of leukemia cells are critically dependent on syndecan signaling, as loss of this signal impairs their localization, migration and dissemination to distant sites. Finally, at a molecular level, syndecan loss directly impairs integrin β 7 function, suggesting that syndecan exerts its influence, at least in part, by coordinating integrin activity in bcCML. These data present a platform for delineating the biological underpinnings of leukemia stem cell function, and highlight the Sdc1-Itgβ7 signaling axis as a key regulatory control point for bcCML growth and dissemination.

Poorly differentiated aggressive myeloid diseases such as Acute Myelogenous Leukemia (AML) and blast crisis Chronic Myelogenous Leukemia (bcCML) are often resistant to standard therapy and associated with significantly poor survival in both children and adults. There is thus a significant need for a better understanding of the mechanisms that drive disease progression and for finding novel therapeutic targets. Thus, to determine the molecular effectors of myeloid leukemia growth in vivo, we carried out a genome-wide CRISPR/Cas9 dropout screen using the lentiviral Brie gRNA library. This library targets 19,674 genes, and has on average 3 gRNAs for each gene, and 1000 control non-targeting gRNAs. We carried out this whole-genome screen in a mouse model of Cas9+ blast crisis CML (bcCML) driven by BCR-ABL/ NUP98-HOXA9 since this represents a very aggressive phase of myeloid cancer where 90% of the leukemic blasts are undifferentiated and cancer stem cell-like. This in vivo screen led to the identification of 3636 genes essential for leukemic growth and propagation in the bone marrow of recipient mice, constituting pathways such as metabolism, protein translation and DNA replication. The genes that were significantly depleted included known drivers of myeloid cancer progression and regulators of myeloid cancer stem cells (for example, Brd4, Kdm1a, Pafah1b1/Lis1, Rptor), indicating that our screening strategy can successfully identify functional drivers of cancer growth. While intrinsic signals that drive myeloid cancer progression are well described, little is known about how interactions with the surrounding microenvironment can control leukemic growth and propagation. Our whole-genome screen identified ~130 cell surface genes that are significantly depleted in the bcCML stem cells transplanted in vivo. Since environmental factors commonly signal through receptors on the surface of leukemic cells, this subset is likely to include most, if not all, genetic effectors of niche driven signals required for in vivo growth and propagation of aggressive myeloid leukemia cells. Of these 130 genes, several have earlier been shown by us and others to be essential for myeloid cancer progression including Itgb1, Cxcr4 and Cd44. We are currently testing the functional contribution of novel candidate cell surface molecules, which can integrate signals from the environment, on the in vivo growth and progression of myeloid malignancies. We anticipate that these studies will provide a basis for testing antibody-mediated therapeutic inhibition of specific microenvironmental signals on myeloid leukemia growth and propagation. No relevant conflicts of interest to declare.

Intratumoral heterogeneity is a common feature of many myeloid leukemias and a significant reason for treatment failure and relapse. Thus identifying the cells responsible for residual disease and leukemia re-growth is critical to better understand how they are regulated. Here we show that a knock-in reporter mouse for the stem cell gene Musashi 2 (Msi2) allows identification of therapy resistant leukemia propagating cells in aggressive myeloid malignancies, and provides a new strategy for defining their core dependencies. Specifically, we carried out a high throughput screen using Msi2 reporter blast crisis chronic myeloid leukemia (bcCML) and identified syndecan-1 (Sdc1), a cell surface proteoglycan, as preferentially expressed in therapy resistant bcCML cells, and critical for bcCML function. Specifically, in Sdc1-/- mice, Sdc1 loss led to a defect in bcCML growth and propagation in vitro and in vivo, and markedly improved survival. Further, live imaging revealed that Sdc1 loss had a striking impact on the spatiotemporal dynamics of leukemia cells, impairing their localization, migration and systemic dissemination. Mechanistically, distinct elements of Sdc1 contributed to leukemia growth and dissemination, with the core protein alone being able to rescue the growth defect, but the heparin sulfate chains that mediate matrix attachment being needed for migration. These data present a new platform for delineating the biological underpinnings of leukemia stem cell function, and identify Sdc1 as a central regulator of leukemia stem cell growth and dissemination.

Synergistic cues from extracellular matrix and soluble factors are often obscure in differentiation. Here the rigidity of cross-linked collagen synergizes with retinoids in the osteogenesis of human marrow mesenchymal stem cells (MSCs). Collagen nanofilms serve as a model matrix that MSCs can easily deform unless the film is enzymatically cross-linked, which promotes the spreading of cells and the stiffening of nuclei as both actomyosin assembly and nucleoskeletal lamin-A increase. Expression of lamin-A is known to be controlled by retinoic acid receptor (RAR) transcription factors, but soft matrix prevents any response to any retinoids. Rigid matrix is needed to induce rapid nuclear accumulation of the RARG isoform and for RARG-specific antagonist to increase or maintain expression of lamin-A as well as for RARG-agonist to repress expression. A progerin allele of lamin-A is regulated in the same manner in iPSC-derived MSCs. Rigid matrices are further required for eventual expression of osteogenic markers, and RARG-antagonist strongly drives lamin-A-dependent osteogenesis on rigid substrates, with pretreated xenografts calcifying in vivo to a similar extent as native bone. Proteomics-detected targets of mechanosensitive lamin-A and retinoids underscore the convergent synergy of insoluble and soluble cues in differentiation.

Filoviruses such as Ebola are microns long but biophysical advantages for such encapsulating/enveloped viruses have remained obscure. Flexible 'filomicelles' have been made from amphiphilic block copolymers and demonstrate effective delivery of two very different hydrophobic compounds. Retinoic acid (RA) and other retinoids regulate RA receptor transcription factors that induce differentiation and arrest proliferation of many cell types, including cancer cells. Lamin-A is transcriptionally regulated by RA receptors, and as a structural protein surrounding chromatin, lamin-A can affect differentiation and karyokinesis as well as nuclear viscosity. Paclitaxel, on the other hand, stabilizes microtubules and induces aneuploidy by blocking mitosis at the metaphase-anaphase transition, which greatly increases cell death. When cancer cells are treated with either of the drugs alone over several periods of the normal cell cycle, cancer cell populations revert back to the original proliferative state, consistent with relapse commonly seen after conventional chemotherapy. On the other hand, combining RA with select chemotherapeutics has for several decades produced durable cures of select cancers, notably pro-myeloblastic leukemia (PML) where RA differentiates cells while chemotherapeutic kills the cancer stem cell. With carcinoma lines, we find dual treatment with RA plus Paclitaxel increases lamin-A levels, aneuploidy, and cell death beyond those achieved by either drug single-handedly, with effects appearing irreversible. Trends with the key cell cycle factor Cyclin-D1 and proliferation marker Ki-67 help clarify the basis for drug synergy. These effects are greatly enhanced by loading the drugs into filomicelles self-assembled from degradable di-block copolymers of Polyethylene glycol-Polybenzyl caprolactone (PEG-PBCL). Preliminary tests in vivo demonstrate sustained delivery for days as well as efficacy in shrinking tumors. These results highlight the irreversible synergy of killing cancerous cells while driving differentiation.

Cell-based immunotherapies such as those based on engineered T-cells appear safe and often effective against liquid tumors. In solid tumors, macrophages are typically abundant, but the density of tumor associated macrophages (TAMs) correlates with poor clinical outcomes as they promote tumor growth and immunosuppression. In our studies, less differentiated donor marrow phagocytes are engineered in order to target tumors and selectively phagocytose cancer cells. Xenograft tumors were first made on the flanks of NSG mice using a tdTomato human lung carcinoma cell line (A549). Systemic injections of anti-human IgG (anti-hum) with large tumors (~70 mm2) showed no effect on tumor growth. However, systemic injection of bone marrow from donor NSG mice together with biweekly anti-hum treatments effectively stopped growth of the solid tumors. Replacing anti-hum with a non-specific antibody had no effect on tumor growth. Based on tdTomato signal intensity within macrophages isolated from tumors, 5-10-fold more donor macrophages are phagocytic compared to resident TAMs (2-3% are phagocytic). Since cancer cells express on their surface 'self' markers that limit the phagocytosis of these cells, we inhibited the 'self' receptors on the injected donor phagocytes prior to systemic injection of the donor marrow. This combination of 'self'-receptor inhibition with anti-hum causes a rapid decrease in tumor burden, shrinking tumors by ~40% in just 10 days compared to a similar growth of untreated tumors in the same time period. The anti-hum injection was again necessary as injection of a non-specific antibody failed to affect tumor growth. Tumor analysis showed that >65% of macrophages that were 'self'-receptor inhibited had phagocytosed the tdTomato A549 cells, which is ~20-fold greater than resident macrophages. Importantly, these cell therapy treatments appear safe with no significant decreases in hematocrit or platelets, which is unlike the anemia that has been reported upon systemic injection of 'self' inhibitors. Our results thus suggest that therapies based on engineered macrophages can be safe and effective against solid tumors if three requirements are met: a phagocytic phenotype, target opsonization, and inhibition of 'self' signaling. Future experiments will involve characterizing macrophage phenotypes of donor versus resident macrophages as well as efforts to maintain the necessary phagocytic phenotype.

Integration of soluble factors and physical properties of extracellular matrix is likely key to stem cell differentiation but the extent to which those pathways overlap remains unclear. Here motivated by the micromechanics of osteogenic niche we looked at the synergy between matrix stimuli and pharmacological perturbation of Retinoic Acid (RA) pathway on primary and iPSC-derived mesenchymal stem cells (MSCs) including iPSC- derived cells from progeria patients towards osteogenesis. Retinoic acid receptor RARG transcription factor is known to regulate nucleoskeletal protein Lamin-A. A cell-by-cell analysis showed that rigid matrix favor higher LMNA and correlates with increased nuclear-to-cytoplasmic ratio of RARG. We found that the Progerin allele of lamin-A is similarly regulated by specific RARG agonist/antagonists. A mechanochemical gene circuit in which tension on lamin-A ultimately favors RARG activity describes well the experimentally observed trend. Scatter-plots of single-cell analyses also show that some cells on stiff substrates fall within the response envelopes of cells cultured on soft substrates and that shared sub- population of non-responding cells we also found that don’t respond to RA agonist or antagonist regulation of Lamin-A. RA antagonist drove lamin-A dependent upregulation of osteogenic markers on rigid substrates and pretreated xenografts showed bone-level calcification suggesting a synergistic effect of soluble and insoluble factors on subpopulation of stem cells that are highly mechanoresponsive.

489-Pos Board B269 Matrix and Soluble Factor Pathways to Lineage Specification Irena L. Ivanovska, Joe Swift, Kyle Spinler, Dave Dingal, Dennis E. Discher. University of Pennsylvania, Philadelphia, PA, USA. Integration of soluble factors and physical properties of extracellular matrix is likely key to stem cell differentiation but the extent to which those pathways overlap remains unclear. Here motivated by the micromechanics of osteogenic niche we looked at the synergy between matrix stimuli and pharmacological perturbation of Retinoic Acid (RA) pathway on primary and iPSC-derived mesenchymal stem cells (MSCs) including iPSCderived cells from progeria patients towards osteogenesis. Retinoic acid receptor RARG transcription factor is known to regulate nucleoskeletal protein Lamin-A. A cell-by-cell analysis showed that rigid matrix favor higher LMNA and correlates with increased nuclear-to-cytoplasmic ratio of RARG. We found that the Progerin allele of lamin-A is similarly regulated by specific RARG agonist/antagonists. A mechanochemical gene circuit in which tension on lamin-A ultimately favors RARG activity describes well the experimentally observed trend. Scatter-plots of single-cell analyses also show that some cells on stiff substrates fall within the response envelopes of cells cultured on soft substrates and that shared subpopulation of non-responding cells we also found that don’t respond to RA agonist or antagonist regulation of Lamin-A. RA antagonist drove laminA dependent upregulation of osteogenic markers on rigid substrates and pretreated xenografts showed bone-level calcification suggesting a synergistic effect of soluble and insoluble factors on subpopulation of stem cells that are highly mechanoresponsive.

AIM:In order to improve the delivery of aromatic drugs by micellar assemblies, and particularly by long and flexible filomicelles, aromatic groups were integrated into the hydrophobic block of a degradable diblock copolymer.MATERIALS & METHODS:Aromatic filomicelles were formed by self-directed assembly of amphiphilic diblock copolymer PEG-PBCL with suitable block ratios. Worm-like filomicelles with an aromatic core were loaded with a common chemotherapeutic, Paclitaxel, for tests of release as well as effects on cancer cell lines in vitro and in vivo.RESULTS:Aromatic filomicelles loaded more Paclitaxel than analogous aliphatic systems. Cell death and aneuploidy of surviving cells (which indicates toxicity) were highest for carcinoma lines treated in vitro with the new filomicelles. Initial tests in vivo also suggest more potent tumor shrinkage.CONCLUSION:Flexible filomicelles with an aromatic core form an efficient drug delivery system that leads to higher cell death than previously reported systems, while inducing aneuploidy in surviving cells.

Megakaryocyte ploidy and the generation of pre/proplatelets are both increased in culture by pharmacologic inhibition of myosin-II, but nonmuscle myosin-IIA (MIIA) mutations paradoxically cause MYH9-related diseases (MYH9-RD) that adversely affect platelets. In marrow, megakaryocytes extend projections into the microcirculation, where shear facilitates fragmentation to large pre/proplatelets, suggesting that fluid stresses and myosin-II activity somehow couple in platelet biogenesis. Here, in bulk shear, plateletlike particles generated from megakaryocytes are maximized at a shear stress typical of that in the microcirculation and after treatment with a myosin-II inhibitor. MIIA activity in static cells is naturally repressed through phosphorylation at Serine-1943, but shear decreases phosphorylation, consistent with MIIA activation and localization to platelet cortex. Micropipette aspiration of cells shows myosin-II accumulates at stressed sites, but its inhibition prevents such mechanoactivation and facilitates generation of CD41(+) fragments similar in size to pre/proplatelets. MYH9-RD mutants phenocopy inhibition, revealing a dominant negative effect. MIIA is diffuse in the large platelets of a MYH9-RD patient with macrothrombocytopenia and is also diffuse in normal pre/proplatelets treated with inhibitor that blocks in vitro division to small platelets. The findings explain the large platelets in MYH9-RD and the near-normal thrombocrit of patients. Myosin-II regulation thus controls platelet size and number.

Professional phagocytes of the mononuclear phagocyte system (MPS), especially ubiquitous macrophages, are commonly thought to engulf or not a target based strictly on 'eat me' molecules such as Antibodies. The target might be a viable 'self' cell or a drug-delivering nanoparticle, or it might be a cancer cell or a microbe. 'Marker of Self' CD47 signals into a macrophage to inhibit the acto-myosin cytoskeleton that makes engulfment efficient. In adhesion of any cell, the same machinery is generally activated by rigidity of target surfaces, and recent results confirm phagocytosis is likewise driven by the rigidity typical of microbes and many synthetics. Basic insights are already being applied in order to make macrophages eat cancer or to delay nanoparticle clearance for better drug delivery and imaging.

Stem cell differentiation is regulated by both soluble factors and the physical properties of extracellular matrix, but the extent to which differentiation pathways are distinct or overlap is often unclear. Here, the micromechanical stiffness of the collagenous bone surface together with broad compositional correlations with collagen-I across many soft tissues suggests enzymatic cross-linking of matrix correlates with nucleoskeletal protein lamin-A, with a retinoid receptor RARG, and with induction toward osteogenesis. Collagen films just 2 nm thick on mica were stiffened or not by transglutaminase cross-linking and used as minimal culture substrates for Mesenchymal stem cells (MSCs). Cells pulling on pristine nano-films visibly deformed and aligned with the collagen fibrils, but on cross-linked films, cells spread isotropically as if adhering to a substrate of greater effective stiffness. Cell nuclei also spread and stiffened, with an increase of lamin-A, nuclear localization of RARG, and upregulation of key early and late osteogenic factors. RARG antagonists also increased lamin-A, and enhanced osteogenesis on rigid substrates in vitro as well as in xenografts of MSCs in mice. A model of the underlying Mechanochemical Gene Circuit couples the sensitivity of stem cells to both insoluble and soluble factors, while a proteomic comparison underscores both differences and overlaps in differentiation pathways.

Tissue microenvironments are characterized not only in terms of chemical composition but also by collective properties such as stiffness, which influences the contractility of a cell, its adherent morphology, and even differentiation. The nucleoskeletal protein lamin-A,C increases with matrix stiffness, confers nuclear mechanical properties, and influences differentiation of mesenchymal stem cells (MSCs), whereas B-type lamins remain relatively constant. Here we show in single-cell analyses that matrix stiffness couples to myosin-II activity to promote lamin-A,C dephosphorylation at Ser22, which regulates turnover, lamina physical properties, and actomyosin expression. Lamin-A,C phosphorylation is low in interphase versus dividing cells, and its levels rise with states of nuclear rounding in which myosin-II generates little to no tension. Phosphorylated lamin-A,C localizes to nucleoplasm, and phosphorylation is enriched on lamin-A,C fragments and is suppressed by a cyclin-dependent kinase (CDK) inhibitor. Lamin-A,C knockdown in primary MSCs suppresses transcripts predominantly among actomyosin genes, especially in the serum response factor (SRF) pathway. Levels of myosin-IIA thus parallel levels of lamin-A,C, with phosphosite mutants revealing a key role for phosphoregulation. In modeling the system as a parsimonious gene circuit, we show that tension-dependent stabilization of lamin-A,C and myosin-IIA can suitably couple nuclear and cell morphology downstream of matrix mechanics.

Self-renewal and differentiation of stem cells depend on asymmetric division and polarized motility processes that in other cell types are modulated by nonmuscle myosin-II (MII) forces and matrix mechanics. Here, mass spectrometry-calibrated intracellular flow cytometry of human hematopoiesis reveals MIIB to be a major isoform that is strongly polarized in hematopoietic stem cells and progenitors (HSC/Ps) and thereby downregulated in differentiated cells via asymmetric division. MIIA is constitutive and activated by dephosphorylation during cytokine-triggered differentiation of cells grown on stiff, endosteum-like matrix, but not soft, marrow-like matrix. In vivo, MIIB is required for generation of blood, while MIIA is required for sustained HSC/P engraftment. Reversible inhibition of both isoforms in culture with blebbistatin enriches for long-term hematopoietic multilineage reconstituting cells by 5-fold or more as assessed in vivo. Megakaryocytes also become more polyploid, producing 4-fold more platelets. MII is thus a multifunctional node in polarized division and niche sensing.

Megakaryocytes (MKs) in the marrow extend projections into blood flow and generate platelets under shear. Understanding MK differentiation and platelet production is of broad clinical importance and extends a need to augment platelet numbers in patients. Reversible but sustained inhibition of non-muscle myosin-II (NMM-II) with the drug blebbistatin increases MK polyploidization, proplatelet formation, and membrane flexibility, thereby increasing platelet generation under shear. Using a cone and plate rheometer to apply fluid shear to drug-treated MKs in bulk, platelet-like-particles (PLPs) that are collagen-I responsive can be generated with intermediate shear. The MKs naturally down-regulate NMM-IIA activity through phosphorylation of S1943, but this site proves shear sensitive, consistent with results for human platelets. Using micropipette aspiration of MKs, inhibition of NMM-IIA is found necessary to generate CD41+ fragments that approximate the size of human platelets. Localization of NMM-IIA to the fragments is modulated by S1943 as seen by unique distribution patterns resulting from specific S1943 mutations that can be abrogated by addition of blebbistatin. The approach is extended to clinically relevant mutations associated with May-Hegglin anomaly (MHA) co-expressed with wild type protein to mimic heterozygotes. As with blebbistatin inhibition of myosin, May-Hegglin mutants result in a higher frequency of fragmentation during micropipette aspiration, indicating a dominant negative effect. Immunofluorescence documents abnormal myosin aggregation in cells transfected with May-Hegglin myosin mutations compared to wild type constructs. Finally, peripheral blood from a patient with a D1414N May-Hegglin mutation is cultured to produce megakaryocytes used to support both the micropipette and immunofluorescence results. These findings reveal a phospho-switch in NMM-II, from inactive to active in the terminal stages of platelet-poiesis, and that proper myosin activity is critical to fragment size and number. Disruption of normal activity enhances fragment generation suggesting a novel mechanism in MHA: in particular, MHA thrombocytopenia results in an increased thrombocrit due to abnormally large platelets, which overcompensates for the reduction in platelet number.

Abstract Abstract 3456 Megakaryocytes (MKs) are rare cells that generate about 1010 platelets every day, which are necessary for vascular homeostasis via clot formation and contraction. Understanding MK differentiation and platelet production is of broad clinical importance and extends to a need to augment platelet numbers in patients. Our group has demonstrated that reversible but sustained inhibition of non-muscle myosin-II (NMM-II) increases MK polyploidization, proplatelet formation, and membrane flexibility, thereby increasing platelet generation (Shin, Spinler, et al., PNAS, 2011; 108:11458–63). The terminal steps of hematopoiesis involve the coordinated maturation and migration of multipotent hematopoietic stem cells (HSCs) from the bone marrow to the perivascular niche. Bulky, polyploid MKs do not easily transmigrate into blood, but do extend tubular membrane ‘proplatelet’ projections into the bloodstream. Visualization by others has shown that blood shear stress fragments the projections into circulating platelets, motivating a controlled study of shear effects on MKs in vitro. To recapitulate this environment in vitro, a cone and plate rheometer is used to impart physiologically relevant shear stress on MEG01s, a human MK cell line. These experiments reveal that platelet-like-particle (PLP) generation is sensitive to both biomechanical and pharmacological factors namely blebbistatin inhibition of NMM-II. We demonstrate that shear stress reduces phosphor-deactivation of NMM-II heavy chain at Ser1943 to approximately 30% of the unsheared level, which indicates a restoration of NMM-II activity necessary for proper platelet function. Stimulation of rheometer generated PLP cultures with collagen-I showed aggregation and phosphatidylserine exposure (with Annexin-V binding in the presence of Ca2+). These data demonstrates that PLPs generated in this system retain some degree of functionality such that MKs exposed to shear stress and blebbistatin result in approximately 6.5 fold more PLPs than untreated MK cultures. Other groups have shown the transport and assembly of platelet organelles to be microtubule dependent and occur de novo along proplatelets (Italiano, et al, Blood, 2005; 106:4066–75). To assess partitioning and segregation of proteins from MKs in sheared membranes, we used fluorescence-imaged micro-deformation (FIMD) to monitor CD41 and NMM-IIA during micropipette aspiration. Studies of erythrocytes had already shown a rich variety of membrane component responses to membrane distension (Discher, et al, Science, 1994; 266:1032–5), but the methods have not yet been applied to MKs. Antibody labeling of MK surface CD41 shows a homogeneous intensity along the aspirated projection of membrane, but cell body shows approximately 3 fold higher intensity, suggesting an excess of CD41 in the MK. Pre-treatment with blebbistatin increases fragmentation frequency, and these fragments show a similar trend with CD41 expression. Nucleofection was used to introduce either GFP tagged WT NMM-IIA or phosphomemetic, myosin deactivating, GFP tagged NMM-IIA S1943D to assess whether pSer impacts partitioning of this cytoskeletal protein that is abundant in platelets. Both WT and S1943D NMM-IIA are seen in the aspirated cell projection, but WT NMM-IIA clearly accumulates at the leading edge of the aspirated projection and at sites of membrane fission and fragmentation, whereas S1943D remains uniformly dispersed. These findings thus underscore the central role that NMM-II heavy chain phosphorylation, and thus activity, in proplatelet formation and platelet fragmentation. Disclosures: No relevant conflicts of interest to declare.

Chemical triggering of membrane domain dynamics is of broad relevance to cell signaling through lipid bilayers and might also be exploited in application of phase-separated vesicles. Here we describe the morphodynamics and remixing kinetics of spotted polymersomes made with mixtures of polyanionic and neutral amphiphiles plus calcium. Addition of the calcium chelator EDTA to vesicle dispersions produced a decrease in domain size within minutes, whereas increasing the pH with NaOH led to the viscous fingering of domains and decreased domain size over hours. Although the latter suggests that the charge of the polyanion contributes to domain formation, the remixing of more negative chains at high pH is surprising. Domain roughening at high pH is also accelerated by EDTA, which highlights the dominance of cross-bridging. Importantly, even though vesicles were perturbed only externally, the inner and outer leaflets remain coupled throughout, consistent with molecular dynamics simulations and suggestive of an order-disorder transition that underlies the remixing kinetics.