M-Phase oscillations in PP1 activity are essential for accurate progression through mammalian oocyte meiosis

Tightly controlled fluctuations in kinase and phosphatase activity play important roles in regulating M-Phase transitions (e.g., G2/M). Protein Phosphatase 1 (PP1) is one of these phosphatases, with oscillations in activity driving mitotic M-Phase entry, progression, and exit, with evidence from a variety of experimental systems pointing to roles in meiosis as well. Here we report that PP1 is important for M-Phase transitions through mouse oocyte meiosis. Employing a unique small-molecule approach to inhibit or activate PP1 at distinct phases of mouse oocyte meiosis, we found that aberrations in normal cyclical PP1 activity leads to meiotic abnormalities. We report here that temporal control of PP1 activity is essential for G2/M transition, metaphase I/anaphase I transition, and the formation of a normal metaphase II oocyte. Our data also reveal that inappropriate activation of PP1 is more deleterious at G2/M transition than at prometaphase I-to-metaphase I, and that an active pool of PP1 during prometaphase I is vital for metaphase I/anaphase I transition and metaphase II chromosome alignment. Taken together, these results establish that loss of oscillations in PP1 activity causes a range of severe meiotic defects, pointing to essential roles for PP1 in oocytes and female fertility, and more broadly, M-Phase regulation.

The size and the age of the metabolically active carbon in tree roots

Little is known about the sources and age of C respired from tree roots. Previous research in tree stems has identified two functional pools of non-structural carbohydrates (NSC): an ‘active’ pool supplied directly from canopy photo-assimilates that supports metabolism and a ‘stored’ pool used when fresh C supplies are limited. We compared the C isotope composition of water soluble NSC and respired CO for aspen roots (Populus tremula hybrids) that were cut off fresh C supply via stem-girdling and prolonged incubation of excised roots. We used bomb radiocarbon to estimate the time elapsed since C fixation for respired CO, water-soluble C, and structural α-cellulose. While freshly excised roots respired CO with mean age <1 yr, within a week the age increased to 1.6-2.9 yr. Freshly excised roots from trees girdled ~3 months previously had similar respiration rates and NSC stocks as un-girdled trees, but respired older C (~1.2 yr). We estimate the NSC in girdled roots must be replaced 5-7 times by reserves remobilized from root-external sources. Using a mixing model and observed correlations between ΔC of water-soluble C and α-cellulose, we estimate ~30% of C is ‘active’ (~5 mg C g).

Myosin-X FERM domain modulates integrin activity at filopodia tips

Filopodia assemble unique integrin-adhesion complexes as they sense and attach to the surrounding extracellular matrix. Integrin activation is essential for filopodia stability; however, the regulation of integrin activity within filopodia is poorly defined. Using structured illumination and scanning electron microscopy, we observed that active integrin is spatially confined to filopodia tips and inactive integrin localises throughout the filopodia shaft. RNAi depletion of integrin regulators validated FERM domain containing talin and MYO10 as critical regulators of filopodia function. Importantly, deletion of MYO10-FERM ablates the active pool of integrin from filopodia, indicating that MYO10 FERM domain is required for integrin activation but not for integrin transport to filopodia tips. Yet, remarkably, the MYO10-FERM domain binds both and β integrin tails restricting integrin activation. Swapping MYO10-FERM with talin-FERM leads to an over-activation of integrin receptors in filopodia. Our observations demonstrate a complex regulation of integrin activity, at filopodia tips, via MYO10-FERM domain and challenge the concept of MYO10-dependent integrin transport in filopodia.

Prevalence of the Hippo Effectors YAP1/TAZ in Tumors of Soft Tissue and Bone

Tumors of soft tissue and bone represent a heterogeneous group of neoplasias characterized by a wide variety of genetic aberrations. Albeit knowledge on tumorigenesis in mesenchymal tumors is continuously increasing, specific insights on altered signaling pathways as a basis for molecularly targeted therapeutic strategies are still sparse. The aim of this study was to determine the involvement of YAP1/TAZ-mediated signals in tumors of soft tissue and bone. Expression levels of YAP1 and TAZ were analyzed by immunohistochemistry in a large cohort of 486 tumor specimens, comprising angiosarcomas (AS), Ewing sarcomas, leiomyosarcomas, malignant peripheral nerve sheath tumors (MPNST), solitary fibrous tumors, synovial sarcomas (SySa), well-differentiated/dedifferentiated/pleomorphic and myxoid liposarcomas (MLS). Moderate to strong nuclear staining of YAP1 and TAZ was detected in 53% and 33%, respectively. YAP1 nuclear expression was most prevalent in MPNST, SySa and MLS, whereas nuclear TAZ was predominately detected in AS, MLS and MPNST. In a set of sarcoma cell lines, immunoblotting confirmed nuclear localization of YAP1 and TAZ, corresponding to their transcriptionally active pool. Suppression of YAP1/TAZ-TEAD mediated transcriptional activity significantly impaired sarcoma cell viability in vitro and in vivo. Our findings identify nuclear YAP1 and TAZ positivity as a common feature in subsets of sarcomas of soft tissue and bone and provide evidence of YAP1/TAZ-TEAD signaling as a specific liability to be considered as a new target for therapeutic intervention. Nuclear YAP1/TAZ expression may represent a biomarker suited to identify patients that could benefit from YAP1/TAZ-TEAD directed therapeutic approaches within future clinical trials.

Arp2/3-Branched Actin Maintains an Active Pool of GTP-RhoA and Controls RhoA Abundance

Small GTPases regulate cytoskeletal dynamics, cell motility, and division under precise spatiotemporal control. Different small GTPases exhibit cross talks to exert feedback response or to act in concert during signal transduction. However, whether and how specific cytoskeletal components’ feedback to upstream signaling factors remains largely elusive. Here, we report an intriguing finding that disruption of the Arp2/3-branched actin specifically reduces RhoA activity but upregulates its total protein abundance. We further dissect the mechanisms underlying these circumstances and identify the altered cortactin/p190RhoGAP interaction and weakened CCM2/Smurf1 binding to be involved in GTP-RhoA reduction and total RhoA increase, respectively. Moreover, we find that cytokinesis defects induced by Arp2/3 inhibition can be rescued by activating RhoA. Our study reveals an intricate feedback from the actin cytoskeleton to the small GTPase. Our work highlights the role of Arp2/3-branched actin in signal transduction aside from its function in serving as critical cytoskeletal components to maintain cell morphology and motility.

Temperature sensitivity of SOM decomposition governed by aggregate protection and microbial communities

Temperature sensitivity (Q10) of soil organic matter (SOM) decomposition is a crucial parameter for predicting the fate of soil carbon (C) under global warming. However, our understanding of its regulatory mechanisms remains inadequate, which constrains its accurate parameterization in Earth system models and induces large uncertainties in predicting terrestrial C-climate feedback. Here, we conducted a long-term laboratory incubation combined with a two-pool model and manipulative experiments to examine potential mechanisms underlying the depth-associated Q10 variations in active and slow soil C pools. We found that lower microbial abundance and stronger aggregate protection were coexisting mechanisms underlying the lower Q10 in the subsoil. Of them, microbial communities were the main determinant of Q10 in the active pool, whereas aggregate protection exerted more important control in the slow pool. These results highlight the crucial role of soil C stabilization mechanisms in regulating temperature response of SOM decomposition, potentially attenuating the terrestrial C-climate feedback.

REHABILITATION AND KINESIOTHERAPEUTIC TREATMENT DURING SURGERY FOR HUMERUS FRACTURE WITH PSEUDOARTHROSIS – A CASE REPORT

The bone of the upper arm (humerus) is the third largest and strongest bone in the human body which is located between the shoulder joint and the elbow joint.The aim of this thesis is to present a multifragmental fracture of the humerus of a 69 year old woman with no neurovascular lesions, with nounion which was treated with an osteosynthesis-adapted plate and individually placed screws. With appropriate physical therapy, the patient has fully recovered and has successfully returned to her everyday activities.Material and methods After the surgical procedure, the 69 year old patient received treatment at the department for physical therapy within GOB 8-mi Septemvri in Skopje. As part of the physical therapy, she received electrotherapy with an IF current and magnetic therapy, kinesiotherapy with active and active assisted exercise, including hydrotherapy with active pool exercises. The success is measured in milimeteres, which often makes this procedure extremely hard for the patient and the therapist.Results The success of the physical treatment of the patient was shown month by month, namely in the first, second and third month of the treatment.Conclusion The Humerus bone must be strong enough to bear the load during lifting, pushing or pulling of objects. The bone of the upper arm is very important, because without this bone we would not be able to control or to use the other parts of the arm.

Paracrine and endocrine modes of myostatin action

Myostatin (MSTN) is a secreted signaling molecule that normally acts to limit muscle mass. In adult animals, MSTN is made almost exclusively by skeletal muscle and circulates in the blood. A critical question is whether this circulating MSTN protein can enter the active pool to regulate muscle growth or whether all of the activity of MSTN results from locally produced protein. Here, we addressed this question in mice by using a Cdx2-Cre transgene in conjunction with a conditional Mstn-flox allele to generate mice in which Mstn was targeted in a regionally restricted manner. Specifically, we generated mosaic mice in which MSTN production was eliminated in posteriorly located muscles but not in anteriorly located muscles, resulting in mice in which circulating levels of MSTN were reduced roughly by half. Analysis of posteriorly located vs. anteriorly located muscles of these mice revealed clear differential effects indicative of an important paracrine role for MSTN in regulating muscle mass. Significant, albeit more subtle, effects consistent with an endocrine mode of MSTN action were also seen in these mice. These findings have important implications not only for the understanding of the physiological control of muscle mass but also for therapeutic strategies to target MSTN to treat patients with muscle loss.

Targeting of the Small GTPase Rab6A′ by the Legionella pneumophila Effector LidA

ABSTRACTWhen the bacteriumLegionella pneumophila, the causative agent of Legionnaires' disease, is phagocytosed by alveolar macrophages, it delivers a large number of effector proteins through its Dot/Icm type IV secretion system into the host cell cytosol. Among those proteins is LidA, an effector that interacts with several host GTPases of the Rab family, including Rab6A′, a regulator of retrograde vesicle trafficking within eukaryotic cells. The effect of LidA on Rab6A′ function and the role of Rab6A′ forL. pneumophilagrowth within host cells has been unclear. Here, we show that LidA preferentially binds Rab6A′ in the active GTP-bound conformation. Rab6 binding occurred through the central region of LidA and followed a stoichiometry for LidA and Rab6A′ of 1:2. LidA maintained Rab6A′ in the active conformation by efficiently blocking the hydrolysis of GTP by Rab6A′, even in the presence of cellular GTPase-activating proteins, suggesting that the function of Rab6A′ must be important for efficient intracellular replication ofL. pneumophila. Accordingly, we found that production of constitutively inactive Rab6A′(T27N) but not constitutively active Rab6A′(Q72L) significantly reduced the ability ofL. pneumophilato initiate intracellular replication in human macrophages. Thus, the presence of an active pool of Rab6 within host cells early during infection is required to support efficient intracellular growth ofL. pneumophila.