Septins constitute a group of GTP binding proteins that assemble into homo- and hetero-oligomeric complexes and filaments. These higher order septin structures are thought to function like scaffolds and/or diffusion barriers serving as spatial localizers for many proteins with key roles in cell polarity and cell cycle progression. In this study, we extensively characterized septin interaction partners using yeast two-hybrid and three-hybrid systems in addition to precipitation analyses in platelets. As a result, we identified human hetero-trimeric septin complexes on a large scale, which had been only postulated in the past. In addition, we illustrated roles of SEPT9 that might contribute to hetero-trimeric septin complex formation. SEPT9 can substitute for septins of the SEPT2 group and partially for SEPT7. Mutagenic analyses revealed that mutation of a potential phosphorylation site in SEPT7 (Y318) regulates the interaction with other septins. We identified several septin-septin interactions in platelets suggesting a regulatory role of diverse septin complexes in platelet function.

† These authors contributed equally to this work.The small GTPase Rac1 is involved in multiple cytosolic functions but recent data point out that Rac1 also translocates to the nucleus to regulate signalling pathways that control gene expression and progression through the cell cycle. Here, we identify the nuclear import receptor karyopherin α2 (KPNA2) as a direct interaction partner of Rac1. The C-terminal polybasic region of Rac1 contains a nuclear localization signal (NLS), whereas Rac2 and Rac3 lack a functional NLS and do not bind to KPNA2. The presence of the NLS in Rac1 determines the specificity of the interaction and is a prerequisite for the nuclear import. Although this interaction is independent of the Rac1 GDP/GTP loading, the induction of the translocation requires Rac1 activation. The activation of Rac1 via the cytotoxic necrotizing factor 1 and the concurrent inhibition of its proteasomal degradation are crucial for the nuclear accumulation of Rac1. Conversely, the reduction of KPNA2 expression inhibits the nuclear import of Rac1. For the first time, our results show a direct interaction between Rac1 and KPNA2 and argue for a KPNA2-dependent nuclear import of Rac1. Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis revealed that nuclear Rac1 coimmunoprecipitates with numerous proteins. In the nucleus, Rac1 may participate in a variety of so far uncharacterized processes.

Inherited platelet defects lead to bleeding symptoms of varying severity. Typically, easy bruising, petechiae, epistaxis, and mucocutaneous bleeding are observed in affected patients. The platelet defects are classified into disorders affecting either platelet surface receptors or intracellular organelles of platelets. The latter are represented by platelet storage pool diseases (SPD) which share a defect of platelet granules. Platelet a-granules, d-granules, or both may be affected resulting in the clinical picture of a-SPD (e.g. Gray platelet syndrome, Quebec platelet disorder, arthrogryposis, renal dysfunction, and cholestasis syndrome), d-SPD (e.g. Hermansky-Pudlak syndrome, Chediak-Higashi syndrome, Griscelli syndrome), or ad-SPD (e.g. X-linked thrombocytopenia, Wiskott-Aldrich syndrome). Diagnosis of SPD is very extensive and requires platelet aggregation and flow cytometry analyses with interpretation from a specialist. Many of these disorders share common treatments, however, efficacy can vary between different patients. Therapy regiments with tranexamic acid, DDAVP, activated FVIIa, and platelet transfusions have been published. Stem cell or bone marrow transplantations are preserved for severe defects. Here, we describe the pathophysiology, clinical manifestations, and diagnosis of the major human SPDs.

The bleeding disorder Bernard-Soulier syndrome (BSS) is caused by mutations in the genes coding for the platelet glycoprotein GPIb/IX receptor. The septin SEPT5 is important for active membrane movement such as vesicle trafficking and exocytosis in non-dividing cells (i.e. platelets, neurons). We report on a four-year-old boy with a homozygous deletion comprising not only glycoprotein Ibβ (GP1BB) but also the SEPT5 gene, located 5' to GP1BB. He presented with BSS, cortical dysplasia (polymicrogyria), developmental delay, and platelet secretion defect. The homozygous deletion of GP1BB and SEPT5, which had been identified by PCR analyses, was confirmed by Southern analyses and denaturing HPLC (DHPLC). The parents were heterozygous for this deletion. Absence of GPIbβ and SEPT5 proteins in the patient's platelets was illustrated using transmission electron microscopy. Besides decreased GPIb/IX expression, flow cytometry analyses revealed impaired platelet granule secretion. Because the bleeding disorder was extremely severe, the boy received bone marrow transplantation (BMT) from a HLA-identical unrelated donor. After successful engraftment of BMT, he had no more bleeding episodes. Interestingly, also his mental development improved strikingly after BMT. This report describes for the first time a patient with SEPT5 deficiency presenting with cortical dysplasia (polymicrogyria), developmental delay, and platelet secretion defect.

Multilineage dysplasia was detected in 32 cases (25%), a frequency similar to that reported by Falini, and was associated with a higher proportion of normal karyotype (93% vs 60%; P Ͻ .001), lower leukocyte count at diagnosis (32 ϫ 10 9 /L vs 69 ϫ 10 9 /L; P ϭ .01), and lower bone marrow infiltration (51% vs 72% blast cells, P Ͻ .001). Interestingly, the frequency of NPM1 and FLT3 internal tandem duplication (FLT3-ITD) mutations did not differ between patients with and without MLD (59% vs 50%, and 31% vs 38%, respectively). NPM1 mutations were found in 68 patients (52%). MLD was observed in 19 patients (28%) with mutated NPM1 and in 13 (21%) with wild-type NPM1. Outcomes in patients with mutated NPM1 were similar for those with and without MLD; response rate was 95% and 85%, 5-year relapse incidence was 35% Ϯ 26% and 47% Ϯ 16%, and 5-year survival was 56% Ϯ 23% and 46% Ϯ 14%, respectively. In contrast in patients with wildtype NPM1, those patients with MLD showed an inferior response rate to induction chemotherapy (53% vs 85%; P ϭ .02). When the analysis was restricted to younger patients (Յ 60 years) those with MLD showed a lower 5-year survival (0% vs 40% Ϯ 16%, P ϭ .012; Figure 1). The unfavorable prognostic value of MLD on response rate (P ϭ .034; relative risk, 4.8; 95% confidence interval, 1.1-20) and survival (P ϭ .036; hazard ratio ϭ 2.5; 95% confidence interval, 1.1-6) was confirmed in a multivariate analysis.These results confirm that, although dysplastic features are a common trait in NPM1-mutated AML, they do not confer a worse prognosis. Falini et al found that gene expression profiling did not identify any distinctive MLD-associated gene signature in the mutated NPM1 cohort. 6 The correlation found in the present study between an unfavorable outcome and dysplastic features in wildtype NPM1 IR-AML patients leads us to suggest that a search for novel genetic or epigenetic markers in this AML subgroup might reveal a specific biologic identity.In conclusion, the prognostic relevance of MLD in IR-AML might be dependent on NPM1 mutational status. Whereas MLD predicts an adverse outcome in patients with wild-type NPM1, it lacks prognostic value in NPM1-mutated AML. Nonetheless, this observation requires further confirmation in a larger series of patients. Contribution: M.D.-B. updated the database of patients included in the analysis, performed all statistical analysis, and wrote the manuscript; M.R. performed morphologic review of all cases and reviewed the article; M.P. updated the database, performed molecular analysis, and reviewed the article; M.T. and M.C. performed molecular analysis and reviewed the article; J.Ll.A. performed morphologic review of all cases and reviewed the article; and J.E. designed the study, supervised statistical analysis, and reviewed the manuscript. Marina Díaz-Beyá Conflict-of-interest disclosure:The authors declare no competing financial interests.

Septins are a novel family of GTP-binding proteins which are essential for cytokinesis, vesicle trafficking, cytoskeletal reorganization and membrane dynamics. They are abundantly expressed in many mitotic cells. Interestingly, they are also expressed in non-dividing cells such as neurons and platelets in which they play an important role in exocytosis. Platelets from SEPT5 knockout mice show an enhanced serotonin secretion and platelet aggregation in response to subthreshold levels of agonists. Septins are associated with a wide array of critical biological events such as neoplasia, neurodegenerative diseases, infections and exocytosis. The role of septins in oncogenesis is complex. Increased expression of some septins seems to trigger the growth of tumor cells. However, other septin isoforms are shown to promote apoptosis and function as tumor suppressor proteins. Interestingly, septins form complexes consisting of multiple septin polypeptides and assemble into filaments and ring-like, higher-order structures. The different septins and their various isoforms seem to determine the function of the septin complex.

Septins are cytoskeletal GTP-binding proteins involved in processes characterized by active membrane movement, such as cytokinesis, vesicle trafficking and exocytosis. Most septins are expressed ubiquitously, however, some septins accumulate in particular tissues. The ubiquitous SEPT11 also shows high expression levels in the central nervous system and in platelets. Here, SEPT11 is involved in vesicle trafficking and may play a role in synaptic connectivity. Interestingly, mice that harbor a homozygous Sept11 null mutation, die in utero. From day 11.5 post coitum onwards, development of homozygous embryos seems to be retarded and the embryos from day 13.5 onwards were dead.

Background: Bernard-Soulier syndrome (BSS) is a severe congenital bleeding disorder characterized by thrombocytopenia, thrombocytopathy and decreased platelet adhesion. BSS results from genetic alterations of the glycoprotein (GP) Ib/IX/V complex. Methods: We report on a patient demonstrating typical BSS phenotype (thrombocytopenia with giant platelets, bleeding symptoms). However, BSS was not diagnosed until he reached the age of 39 years. Results: Flow cytometry of the patient’s platelets revealed absence of GPIb/IX/V receptor surface expression. In addition, immunofluorescence analysis of patient’s platelets demonstrated very faint staining of GPIX. A novel homozygous deletion comprising 11 nucleotides starting at position 1644 of the GPIX gene was identified using molecular genetic analysis. Conclusions: The novel 11-nucleotide deletion (g.1644_1654del11) was identified as causing the bleeding disorder in the BSS patient. This homozygous deletion includes the last 4 nucleotides of the Kozak sequence as well as the start codon and the following 4 nucleotides of the coding sequence. The Kozak sequence is a region indispensable for the initiation of the protein translation process, thus preventing synthesis of functional GPIX protein in the case of deletion.