Spontaneous signal generation by an excitable system for cell migration.

Eukaryotic cells exhibit random migration in the absence of extracellular directional cues. This random migration acts as basal motility for various migratory responses such as chemotaxis. The self-organization of random motility requires the internal signals that determine the anterior side of the migrating cell be generated spontaneously from the intrinsic activities of intracellular signaling networks. Recent studies have identified an excitable system as the mechanism of the spontaneous signal generation. Here, we discuss how the excitable system of Ras, a small G protein, regulates signaling networks in Dictyostelium discoideum as a model organism. The excitability produces a domain where an active form of Ras is enriched on the cell membrane without extracellular directional cues, such that Ras serves as the anterior signal. The typical spatiotemporal characteristics are mathematically explained by reaction-diffusion models. These models further enable a quantitative analysis of the dynamics that depends on the internal cellular states and surrounding environments. Downstream of the Ras excitable system, a phosphoinositide metabolic network composed of PI3K, PTEN, PI(3,4,5)P3 and PI(4,5)P2 exhibits bistability to discretize the anterior and posterior regions of the cell membrane. Upstream, a local excitation and global inhibition local excitation global inhibition network, which works for gradient sensing in the presence of chemoattractant gradients, spatiotemporally biases the excitability of Ras for chemotaxis. In parallel with the Ras excitable system, the cGMP signaling pathway constitutes another excitable system of its own periodicity to ensure flexible migratory dynamics. In addition to these intracellular signaling networks, an intercellular signaling network activated by secreted cAMP is coupled with the Ras excitable system for collective cell migration. Finally, we discuss how the excitable system of Ras operates as a platform of information integration by receiving multiple intrinsic and extrinsic signals to ensure spontaneous cellular activity and robust responses in eukaryotic cell migration under natural complex environments.

A dynamic partitioning mechanism polarizes membrane protein distribution.

The plasma membrane is widely regarded as the hub of the numerous signal transduction activities. Yet, the fundamental biophysical mechanisms that spatiotemporally compartmentalize different classes of membrane proteins remain unclear. Using multimodal live-cell imaging, here we first show that several lipid-anchored membrane proteins are consistently depleted from the membrane regions where the Ras/PI3K/Akt/F-actin network is activated. The dynamic polarization of these proteins does not depend upon the F-actin-based cytoskeletal structures, recurring shuttling between membrane and cytosol, or directed vesicular trafficking. Photoconversion microscopy and single-molecule measurements demonstrate that these lipid-anchored molecules have substantially dissimilar diffusion profiles in different regions of the membrane which enable their selective segregation. When these diffusion coefficients are incorporated into an excitable network-based stochastic reaction-diffusion model, simulations reveal that the altered affinity mediated selective partitioning is sufficient to drive familiar propagating wave patterns. Furthermore, normally uniform integral and lipid-anchored membrane proteins partition successfully when membrane domain-specific peptides are optogenetically recruited to them. We propose "dynamic partitioning" as a new mechanism that can account for large-scale compartmentalization of a wide array of lipid-anchored and integral membrane proteins during various physiological processes where membrane polarizes.

[Prompt cytogenetic response by venetoclax plus azacitidine regimen in a patient with AML harboring double-minute chromosomes with MYC gene amplification].

Double minute chromosomes (dmin) are small, acentric, and extrachromosomal fragments that frequently mediate oncogene amplification and induce rapid disease progression with poor prognosis, although they are infrequent in myeloid neoplasms. An 81-year-old woman with anemia and thrombocytopenia was admitted to our hospital. Bone marrow examination showed 54.0% of the blasts. She was diagnosed with acute myeloid leukemia (French-American-British classification, M2; World Health Organization classification, acute myeloid leukemia [AML], not otherwise specified, AML with maturation). Chromosomal analysis revealed the presence of 3-45 dmin in the background of 46 and XX in 14 out of 20 metaphases examined. Spectral karyotyping examination demonstrated that the dmins were derived from chromosome 8. Fluorescence in situ hybridization (FISH) targeting the MYC gene demonstrated that dmins contained full-length MYC genes with multiple signals. Finally, she was diagnosed with AML with dmin via MYC amplification and was administered with venetoclax plus azacitidine chemotherapy. After two cycles of the regimen, FISH found no MYC amplification signals, indicating her state being in cytogenetic remission. At present, she has finished four cycles of the regimen and remained in complete remission. Venetoclax plus azacitidine could be an effective regimen for the poor prognosis of AML with dmin through MYC amplification.

Sphingomyelin metabolism underlies Ras excitability for efficient cell migration and chemotaxis.

In eukaryotic motile cells, the active Ras (Ras-GTP)-enriched domain is generated in an asymmetric manner on the cell membrane through the excitable dynamics of an intracellular signaling network. This asymmetric Ras signaling regulates pseudopod formation for both spontaneous random migration and chemoattractant-induced directional migration. While membrane lipids, such as sphingomyelin and phosphatidylserine, contribute to Ras signaling in various cell types, whether they are involved in the Ras excitability for cell motility is unknown. Here we report that functional Ras excitability requires the normal metabolism of sphingomyelin for efficient cell motility and chemotaxis. The pharmacological blockade of sphingomyelin metabolism by an acid-sphingomyelinase inhibitor, fendiline, and other inhibitors suppressed the excitable generation of the stable Ras-GTP-enriched domain. The suppressed excitability failed to invoke enough basal motility to achieve directed migration under shallow chemoattractant gradients. The fendiline-induced defects in Ras excitability, motility and stimulation-elicited directionality were due to an accumulation of sphingomyelin on the membrane, which could be recovered by exogenous sphingomyelinase or phosphatidylserine without changing the expression of Ras. These results indicate a novel regulatory mechanism of the excitable system by membrane lipids, in which sphingomyelin metabolism provides a membrane environment to ensure Ras excitation for efficient cellular motility and chemotaxis.Key words: cell polarity, cell migration, Ras, excitability, sphingomyelin.

Clinical implications of NUP98::NSD1 fusion at diagnosis in adult FLT3-ITD positive AML.

OBJECTIVES: The cryptic fusion oncogene NUP98::NSD1 is known to be associated with FLT3-ITD mutation in acute myeloid leukemia (AML), and an independent poor prognostic factor in pediatric AML. However, there are little data regarding the clinical significance of NUP98::NSD1 in adult cohort. METHODS: We conducted a multicenter retrospective study to investigate the prevalence, clinical characteristics, and prognostic impact of NUP98::NSD1 in adult FLT3-ITD-positive AML patients. RESULTS: In a total of 97 FLT3-ITD-positive AML patients, six cases (6.2%) were found to harbor the NUP98::NSD1 fusion transcript. NUP98::NSD1 positive cases had significantly higher platelet counts and a higher frequency of FAB-M4 morphology than NUP98::NSD1 negative cases. NUP98::NSD1 was found to be mutually exclusive with NPM1 mutation, and was accompanied by the WT1 mutation in three of the six cases. The presence of NUP98::NSD1 fusion at the time of diagnosis predicted poor response to cytarabine-anthracycline-based intensive induction chemotherapy (induction failure rate: 83% vs. 36%, p = .038). Five of the six cases with NUP98::NSD1 underwent allogeneic hematopoietic stem cell transplantation (HSCT). Two of the five cases have successfully maintained remission, with one of them being rescued through a second HSCT. CONCLUSIONS: Detecting NUP98::NSD1 in adult FLT3-ITD-positive AML is crucial to recognizing chemotherapy-resistant group.

A dynamic partitioning mechanism polarizes membrane protein distribution.

The plasma membrane is widely regarded as the hub of the signal transduction network activities that drives numerous physiological responses, including cell polarity and migration. Yet, the symmetry breaking process in the membrane, that leads to dynamic compartmentalization of different proteins, remains poorly understood. Using multimodal live-cell imaging, here we first show that multiple endogenous and synthetic lipid-anchored proteins, despite maintaining stable tight association with the inner leaflet of the plasma membrane, were unexpectedly depleted from the membrane domains where the signaling network was spontaneously activated such as in the new protrusions as well as within the propagating ventral waves. Although their asymmetric patterns resembled those of standard peripheral "back" proteins such as PTEN, unlike the latter, these lipidated proteins did not dissociate from the membrane upon global receptor activation. Our experiments not only discounted the possibility of recurrent reversible translocation from membrane to cytosol as it occurs for weakly bound peripheral membrane proteins, but also ruled out the necessity of directed vesicular trafficking and cytoskeletal supramolecular structure-based restrictions in driving these dynamic symmetry breaking events. Selective photoconversion-based protein tracking assays suggested that these asymmetric patterns instead originate from the inherent ability of these membrane proteins to "dynamically partition" into distinct domains within the plane of the membrane. Consistently, single-molecule measurements showed that these lipid-anchored molecules have substantially dissimilar diffusion profiles in different regions of the membrane. When these profiles were incorporated into an excitable network-based stochastic reaction-diffusion model of the system, simulations revealed that our proposed "dynamic partitioning" mechanism is sufficient to give rise to familiar asymmetric propagating wave patterns. Moreover, we demonstrated that normally uniform integral and lipid-anchored membrane proteins in Dictyostelium and mammalian neutrophil cells can be induced to partition spatiotemporally to form polarized patterns, by optogenetically recruiting membrane domain-specific peptides to these proteins. Together, our results indicate "dynamic partitioning" as a new mechanism of plasma membrane organization, that can account for large-scale compartmentalization of a wide array of lipid-anchored and integral membrane proteins in different physiological processes.

Possible Chronic Graft-versus-host Disease in the Central Nervous System Manifesting as Cerebellar Ataxia after Allogeneic Hematopoietic Stem Cell Transplantation for Acute Myeloid Leukemia.

A 44-year-old woman was admitted to our hospital with a fever, dizziness, and gait disturbance after undergoing allogeneic hematopoietic stem cell transplantation for acute myeloid leukemia followed by graft-versus-host disease. She presented with cerebellar ataxia, nystagmus, and numbness of the lower extremities. Brain magnetic resonance imaging and perfusion scintigraphy showed progressive cerebellar involvement. Cerebrospinal fluid tests showed mildly elevated protein and IgG levels without pleocytosis. Anti-ganglioside antibodies were detected, but their levels did not follow the patient's clinical course. The patient did not respond sufficiently to steroids or other immunotherapies. We herein report the clinical characteristics of this case and a literature review.

A sub-population of Dictyostelium discoideum cells shows extremely high sensitivity to cAMP for directional migration.

The chemotaxis of Dictysotelium discoideum cells in response to a chemical gradient of cyclic adenosine 3',5'-monophosphate (cAMP) was studied using a newly designed microfluidic device. The device consists of 800 cell-sized channels in parallel, each 4 µm wide, 5 µm high, and 100 µm long, allowing us to prepare the same chemical gradient in all channels and observe the motility of 500-1000 individual cells simultaneously. The percentage of cells that exhibited directed migration was determined for various cAMP concentrations ranging from 0.1 pM to 10 µM. The results show that chemotaxis was highest at 100 nM cAMP, consistent with previous observations. At concentrations as low as 10 pM, about 16% of cells still exhibited chemotaxis, suggesting that the receptor occupancy of only 6 cAMP molecules/cell can induce chemotaxis in very sensitive cells. At 100 pM cAMP, chemotaxis was suppressed due to the self-production and secretion of intracellular cAMP induced by extracellular cAMP. Overall, systematic observations of a large number of individual cells under the same chemical gradients revealed the heterogeneity of chemotaxis responses in a genetically homogeneous cell population, especially the existence of a sub-population with extremely high sensitivity for chemotaxis.

Single-molecule imaging of PI(4,5)P2 and PTEN in vitro reveals a positive feedback mechanism for PTEN membrane binding.

PTEN, a 3-phosphatase of phosphoinositide, regulates asymmetric PI(3,4,5)P3 signaling for the anterior-posterior polarization and migration of motile cells. PTEN acts through posterior localization on the plasma membrane, but the mechanism for this accumulation is poorly understood. Here we developed an in vitro single-molecule imaging assay with various lipid compositions and use it to demonstrate that the enzymatic product, PI(4,5)P2, stabilizes PTEN's membrane-binding. The dissociation kinetics and lateral mobility of PTEN depended on the PI(4,5)P2 density on artificial lipid bilayers. The basic residues of PTEN were responsible for electrostatic interactions with anionic PI(4,5)P2 and thus the PI(4,5)P2-dependent stabilization. Single-molecule imaging in living Dictyostelium cells revealed that these interactions were indispensable for the stabilization in vivo, which enabled efficient cell migration by accumulating PTEN posteriorly to restrict PI(3,4,5)P3 distribution to the anterior. These results suggest that PI(4,5)P2-mediated positive feedback and PTEN-induced PI(4,5)P2 clustering may be important for anterior-posterior polarization.

Talin B regulates collective cell migration via PI3K signaling in Dictyostelium discoideum mounds.

Collective cell migration is a key process during the development of multicellular organisms, in which the migrations of individual cells are coordinated through chemical guidance and physical contact between cells. Talin has been implicated in mechanical linkage between actin-based motile machinery and adhesion molecules, but how talin contributes to collective cell migration is unclear. Here we show that talin B is involved in chemical coordination between cells for collective cell migration at the multicellular mound stage in the development of Dictyostelium discoideum. From early aggregation to the mound formation, talB-null cells exhibited collective migration normally with cAMP relay. Subsequently, talB-null cells showed developmental arrest at the mound stage, and at the same time, they had impaired collective migration and cAMP relay, while wild-type cells exhibited rotational cell migration continuously in concert with cAMP relay during the mound stage. Genetic suppression of PI3K activity partially restored talB-null phenotypes in collective cell migration and cAMP relay. Overall, our observations suggest that talin B regulates chemical coordination via PI3K-mediated signaling in a stage-specific manner for the multicellular development of Dictyostelium cells.

Myeloid differentiation factor 88 signaling in donor T cells accelerates graft-versus-host disease.

Myeloid differentiation factor 88 (MyD88) signaling has a crucial role in activation of both innate and adoptive immunity. MyD88 transduces signals via Toll-like receptor and interleukin-1 receptor superfamily to the NFκB pathway and inflammasome by forming a molecular complex with interleukin-1 receptor-associated kinase 4. The MyD88/interleukin-1 receptor-associated kinase 4 pathway plays an important role, not only in innate immunity, but also T-cell immunity; however, its role in donor T cells on the pathophysiology of graft-versus-host disease (GvHD) remains to be elucidated. We addressed this issue by using MyD88-deficient T cells in a mouse model of allogeneic hematopoietic stem cell transplantation (allo-SCT). While MyD88-deficient and wild-type T cells proliferated equivalently after transplantation, MyD88-deficient T cells demonstrated impaired survival and differentiation toward Th1, Tc1, and Th17, and induced less severe GvHD compared to wild-type T cells. Administration of interleukin-1 receptor-associated kinase 4 inhibitor PF-06650833 significantly ameliorated GvHD after allo-SCT. These results thus demonstrate that donor T-cell MyD88/interleukin-1 receptor-associated kinase 4 pathway is a novel therapeutic target against GvHD after allo-SCT.

Ibrutinib Caused Mediastinal Emphysema and Pneumothorax in the Treatment of a Patient with Mantle Cell Lymphoma.

A 70-year-old Japanese man with mantle cell lymphoma underwent extensive chemotherapy and radiation because of the relapse of mantle cell lymphoma. He developed mediastinal emphysema and a pneumothorax 14 days after treatment with 560 mg of ibrutinib. The mediastinal emphysema and the right pneumothorax disappeared after the ibrutinib treatment was tapered off. The patient developed interstitial pneumonia without any infection and new lesions of mantle cell lymphoma in the lungs after restarting treatment with 560 mg of ibrutinib. In this case, the patient developed pneumonia after retreatment with ibrutinib, suggesting the small lung fibrosis that penetrated the mediastinum might have caused the emphysema and pneumothorax.

Excitable dynamics of Ras triggers spontaneous symmetry breaking of PIP3 signaling in motile cells.

Spontaneous cell movement is underpinned by an asymmetric distribution of signaling molecules including small G proteins and phosphoinositides on the cell membrane. However, the molecular network necessary for spontaneous symmetry breaking has not been fully elucidated. Here, we report that, in Dictyostelium discoideum, the spatiotemporal dynamics of GTP bound Ras (Ras-GTP) breaks the symmetry due its intrinsic excitability even in the absence of extracellular spatial cues and downstream signaling activities. A stochastic excitation of local and transient Ras activation induced phosphatidylinositol (3,4,5)-trisphosphate (PIP3) accumulation via direct interaction with Phosphoinositide 3-kinase (PI3K), causing tightly coupled traveling waves that propagated along the membrane. Comprehensive phase analysis of the waves of Ras-GTP and PIP3 metabolism-related molecules revealed the network structure of the excitable system including positive-feedback regulation of Ras-GTP by the downstream PIP3. A mathematical model reconstituted a series of the observed symmetry-breaking phenomena, illustrating the essential involvement of Ras excitability in the cellular decision-making process.

Myasthenia gravis after allogeneic bone marrow transplantation: A case report and literature review.

A 52-year-old man with acute myeloid leukemia underwent allogeneic hematopoietic stem cell transplantation and developed extensive chronic graft-versus-host disease and myasthenia gravis (MG), which became involved with oculobulbar and proximal upper and lower limb weakness in 677days. In the literature, we identified 24 cases where MG developed after allo-SCT. Graft-versus-host disease development and male recipients of female donors might be prone to the development of posttransplant MG (odds ratio, 3.75).

[Thrombotic microangiopathy developing subsequent to tocilizumab therapy in a patient with TAFRO syndrome].

We report a case of a 60-year-old male who presented with fever and anasarca as well as hepatosplenomegaly, general lymphadenopathy, and disseminated intravascular coagulation (DIC), and was, therefore, admitted to our hospital. In addition, the patient suffered from respiratory failure and renal dysfunction and had pleural effusion and ascites. The pathological diagnosis from lymph node biopsy suggested multicentric Castleman's disease of the plasma cell type; however, the presence of high IL-6 levels, myelofibrosis, thrombocytopenia, anasarca, renal dysfunction, and hepatosplenomegaly led to a definitive diagnosis of TAFRO syndrome. Tocilizumab was administered on day 15 of disease diagnosis, resulting in the improvement in DIC but not other symptoms. As schizocytes were detected in the peripheral blood, he also experienced disturbance of consciousness and thrombotic microangiopathy (TMA) was considered. Following plasma exchange (PE) and continuous hemodiafiltration (CHDF), his symptoms temporarily improved. However, his condition worsened again, and he eventually died on day 33. Pathological autopsy revealed that although the lymph nodes were not enlarged, he had organomegaly, gastrointestinal and omental hemorrhage, and acute necrotizing pancreatitis. Since TMA developed after the administration of tocilizumab, the possibility of drug-induced secondary TMA cannot be ruled out.

Mutual inhibition between PTEN and PIP3 generates bistability for polarity in motile cells.

Phosphatidylinositol 3,4,5-trisphosphate (PIP3) and PIP3 phosphatase (PTEN) are enriched mutually exclusively on the anterior and posterior membranes of eukaryotic motile cells. However, the mechanism that causes this spatial separation between the two molecules is unknown. Here we develop a method to manipulate PIP3 levels in living cells and used it to show PIP3 suppresses the membrane localization of PTEN. Single-molecule measurements of membrane-association and -dissociation kinetics and of lateral diffusion reveal that PIP3 suppresses the PTEN binding site required for stable PTEN membrane binding. Mutual inhibition between PIP3 and PTEN provides a mechanistic basis for bistability that creates a PIP3-enriched/PTEN-excluded state and a PTEN-enriched/PIP3-excluded state underlying the strict spatial separation between PIP3 and PTEN. The PTEN binding site also mediates the suppression of PTEN membrane localization in chemotactic signaling. These results illustrate that the PIP3-PTEN bistable system underlies a cell's decision-making for directional movement irrespective of the environment.

Stiff-person Syndrome with Waldenström Macroglobulinemia.

We herein report the case of stiff-person syndrome in a 73-year-old woman. She experienced episodes of painful muscle spasms and was admitted to another hospital. She was diagnosed with Waldenström macroglobulinemia. She showed improvement in muscle spasms post-chemotherapy, which was discontinued due to pancytopenia. Six months later, she was admitted to our hospital for repeated whole-body muscle spasms, at which point she was diagnosed with stiff-person syndrome. An anti-glutamic acid decarboxylase antibody text was negative. Her muscle spasms disappeared after the administration of corticosteroids and rituximab. Stiff-person syndrome may develop with Waldenström macroglobulinemia. In the present case, corticosteroids and rituximab provided effective treatment.

Ruxolitinib protects skin stem cells and maintains skin homeostasis in murine graft-versus-host disease.

Graft-versus-host disease (GVHD) is the major complication after allogeneic stem cell transplantation (SCT). Emerging evidence indicates that GVHD leads to injury of intestinal stem cells. However, it remains to be investigated whether skin stem cells could be targeted in skin GVHD. Lgr5+ hair follicle stem cells (HFSCs) contribute to folliculogenesis and have a multipotent capacity to regenerate all epithelial cells in repair. We studied the fate of Lgr5+ HFSCs after SCT and explored the novel treatment to protect Lgr5+ HFSCs against GVHD using murine models of SCT. We found that GVHD reduced Lgr5+ HFSCs in association with impaired hair regeneration and wound healing in the skin after SCT. Topical corticosteroids, a standard of care for a wide range of skin disorders including GVHD, damaged HFSCs and failed to improve skin homeostasis, despite of their anti-inflammatory effects. In contrast, JAK1/2 inhibitor ruxolitinib significantly ameliorated skin GVHD, protected Lgr5+ HFSCs, and restored hair regeneration and wound healing after SCT. We, for the first time, found that GVHD targets Lgr5+ HFSCs and that topical ruxolitinib represents a novel strategy to protect skin stem cells and maintain skin homeostasis in GVHD.

R-Spondin1 expands Paneth cells and prevents dysbiosis induced by graft-versus-host disease.

The intestinal microbial ecosystem is actively regulated by Paneth cell-derived antimicrobial peptides such as α-defensins. Various disorders, including graft-versus-host disease (GVHD), disrupt Paneth cell functions, resulting in unfavorably altered intestinal microbiota (dysbiosis), which further accelerates the underlying diseases. Current strategies to restore the gut ecosystem are bacteriotherapy such as fecal microbiota transplantation and probiotics, and no physiological approach has been developed so far. In this study, we demonstrate a novel approach to restore gut microbial ecology by Wnt agonist R-Spondin1 (R-Spo1) or recombinant α-defensin in mice. R-Spo1 stimulates intestinal stem cells to differentiate to Paneth cells and enhances luminal secretion of α-defensins. Administration of R-Spo1 or recombinant α-defensin prevents GVHD-mediated dysbiosis, thus representing a novel and physiological approach at modifying the gut ecosystem to restore intestinal homeostasis and host-microbiota cross talk toward therapeutic benefits.