Myotubularin-related Proteins 3 and 4 Interact with Polo-like Kinase 1 and Centrosomal Protein of 55 kDa to Ensure Proper Abscission

The myotubularins are a family of phosphatases that dephosphorylate the phosphatidylinositols phosphatidylinositol-3-phosphate and phosphatidylinositol-3,5-phosphate. Several family members are mutated in disease, yet the biological functions of the majority of myotubularins remain unknown. To gain insight into the roles of the individual enzymes, we have used affinity purification coupled to mass spectrometry to identify protein–protein interactions for the myotubularins. The myotubularin interactome comprises 66 high confidence (false discovery rate ≤1%) interactions, including 18 pairwise interactions between individual myotubularins. The results reveal a number of potential signaling contexts for this family of enzymes, including an intriguing, novel role for myotubularin-related protein 3 and myotubularin-related protein 4 in the regulation of abscission, the final step of mitosis in which the membrane bridge remaining between two daughter cells is cleaved. Both depletion and overexpression of either myotubularin-related protein 3 or myotubularin-related protein 4 result in abnormal midbody morphology and cytokinesis failure. Interestingly, myotubularin-related protein 3 and myotubularin-related protein 4 do not exert their effects through lipid regulation at the midbody, but regulate abscission during early mitosis, by interacting with the mitotic kinase polo-like kinase 1, and with centrosomal protein of 55 kDa (CEP55), an important regulator of abscission. Structure-function analysis reveals that, consistent with known intramyotubularin interactions, myotubularin-related protein 3 and myotubularin-related protein 4 interact through their respective coiled coil domains. The interaction between myotubularin-related protein 3 and polo-like kinase 1 relies on the divergent, nonlipid binding Fab1, YOTB, Vac1, and EEA1 domain of myotubularin-related protein 3, and myotubularin-related protein 4 interacts with CEP55 through a short GPPXXXY motif, analogous to endosomal sorting complex required for transport-I components. Disruption of any of these interactions results in abscission failure, by disrupting the proper recruitment of CEP55, and subsequently, of endosomal sorting complex required for transport-I, to the midbody. Our data suggest that myotubularin-related protein 3 and myotubularin-related protein 4 may act as a bridge between CEP55 and polo-like kinase 1, ensuring proper CEP55 phosphorylation and regulating CEP55 recruitment to the midbody. This work provides a novel role for myotubularin-related protein 3/4 heterodimers, and highlights the temporal and spatial complexity of the regulation of cytokinesis.The myotubularins are a subfamily of protein tyrosine phosphatases (PTPs)¹, consisting of sixteen conserved proteins. Despite containing the conserved C(X)₅R catalytic motif found in all protein tyrosine phosphatases, myotubularins harbor active sites that do not dephosphorylate tyrosine, but instead catalyze the conversion of the phosphatidylinositol-type lipids phosphatylinositol 3 phosphate (PI3P) and phosphatylinositol 3,5 phosphate (PI3,5P) to phosphatidylinositol (PI) and phosphatylinositol 5 phosphate (PI5P), respectively (1). Phosphatidylinositols are important molecules in a variety of processes, and as enzymatic regulators, myotubularins may function in cell proliferation, differentiation, survival, and cytoskeletal and junctional dynamics (1, 2). Of the sixteen myotubularins, only nine are active enzymes (supplemental Fig. S1A), as several lack catalytic cysteine residues (3). Myotubularins interact extensively with each other, and interactions between active and inactive pairs are frequent (4). It is thought that inactive myotubularins regulate the activity, substrate binding, and/or localization of their active binding partners (2).Several myotubularins are linked to human disease. Myotubularin (MTM1), the first reported family member, is mutated in X-linked centronuclear myopathy (5), and Myotubularin related protein 14 (MTMR14) is mutated in autosomal centronuclear myopathy (6). Mutations in the active MTMR2 or its inactive binding partner, SET binding factor (SBF)2 (MTMR13), cause Charcot-Marie-Tooth diseases CMT4A and CMT4B, respectively (7–9). MTMR7 and MTMR9 have been associated with metabolic syndrome and obesity (MTMR9) (10, 11), epilepsy (MTMR7/9) (12), and Creutzfeldt-Jakob disease (MTMR7) (13). In addition, misregulation of the active phosphatase MTMR3 contributes to susceptibility to gastric and colon carcinomas (14), oral cancer (15), and lung cancer (16), and contributes to metastasis (15, 17). Aberrant expression of the inactive MTMR11 has been observed in acute myeloid leukemia (18), acute lymphocytic leukemia (19), and Her2-positive breast cancer (20). Generally, myotubularins are thought to integrate different cellular pathways, through both phosphatidylinositol regulation and protein–protein interactions (2). Despite their proposed involvement in a variety of cellular processes as well as disease states, many myotubularins remain poorly characterized, with their precise cellular functions not yet elucidated, and the pathological significance of those functions still unknown.To gain insight on the biological functions of myotubularin family phosphatases, we have used affinity purification coupled to mass spectrometry (AP-MS) to identify protein–protein interactions for each myotubularin. The results expand upon the known repertoire of intra-myotubularin interactions, and, critically, identify specific novel interactions for individual myotubularins, providing valuable clues toward their respective functions. Further investigation revealed an unexpected role for MTMR3 and MTMR4 in abscission (21), the fission event at the end of cytokinesis that severs the final membrane link between divided daughter cells. Future studies of additional identified protein–protein interactions will undoubtedly illuminate the cellular roles of myotubularin family phosphatases.     

A dual biosensor for 2-alkyl-4-quinolone quorum-sensing signal molecules

Pseudomonas, Burkholderia and Alteromonas species produce diverse 2-alkyl-4-quinolones (AHQs) which inhibit the growth of bacteria, algae and phytoplankton, chelate iron, modulate mammalian host immune defences and act as quorum-sensing (QS) signal molecules. To facilitate the detection, identification and quantification of the major Pseudomonas aeruginosa AHQs 2-heptyl-3-hydroxy-4-quinolone (PQS) and 2-heptyl-4-quinolone (HHQ) we developed two different AHQ biosensors. These were constructed by introducing either a lecA::luxCDABE or a pqsA::luxCDABE reporter gene fusion into a P. aeruginosa pqsA mutant which cannot synthesize AHQs. While both biosensors responded similarly to PQS (EC(50) 18 +/- 4 microM), the pqsA::luxCDABE biosensor was most sensitively activated by HHQ (EC(50) 0.44 +/- 0.1 microM). This biosensor was also activated albeit less sensitively by (i) PQS analogues with alkyl chains varying from C1 to C11, (ii) HHQ analogues with C9 and C11 alkyl chains and (iii) 2-heptyl-4-hydroxyquinoline-N-oxide (HHQNO). The AHQ biosensor also responded differentially to the AHQs present in cell free culture supernatants prepared from PAO1 and isogenic strains carrying mutations in genes (pqsA, pqsH, lasR, lasI, rhlR, rhlI) known to influence AHQ production. The AHQ profiles of P. aeruginosa strains was also evaluated by overlaying thin layer chromatogram (TLC) plates with the pqsA::luxCDABE biosensor. In PAO1, three major bioluminescent spots were observed which correspond to PQS, HHQ and a mixture of 2 nonyl-4-quinolone and HHQNO. We also noted that on TLC plates the biosensor not only produced bioluminescence in response to AHQs but also the green pigment, pyocyanin which offers an alternative visual indicator for AHQ production.     

The many faces of actin: matching assembly factors with cellular structures

Actin filaments are major components of at least 15 distinct structures in metazoan cells. These filaments assemble from a common pool of actin monomers, but do so at different times and places, and in response to different stimuli. All of these structures require actin-filament assembly factors. To date, many assembly factors have been identified, including Arp2/3 complex, multiple formin isoforms and spire. Now, a major task is to figure out which factors assemble which actin-based structures. Here, we focus on structures at the plasma membrane, including both sheet-like protrusive structures (such as lamellipodia and ruffles) and finger-like protrusions (such as filopodia and microvilli). Insights gained from studies of adherens junctions and the immunological synapse are also considered.     

Effects of combined administration of captopril and DMSA on arsenite induced oxidative stress and blood and tissue arsenic concentration in rats

We compared the therapeutic efficacy of captopril and a thiol chelating agent, meso 2,3-dimercaptosuccinic acid (DMSA) either individually or in combination against arsenite induced oxidative stress and mobilization of metal in rats. Animals were exposed to 100 ppm arsenite as sodium arsenite in drinking water for six weeks followed by treatment with DMSA (50 mg/kg, orally), captopril (50 mg/kg, intraperitoneally) either alone or in combination, once daily for 5 consecutive days. Arsenite exposure led to a significant depletion of blood delta-aminolevulinic acid dehydratase (ALAD) activity, glutathione and platelet levels while significantly increased the level of reactive oxygen species (in RBCs). Hepatic reduced glutathione (GSH) level showed a significant decrease while, thiobarbituric acid reactive substances (TBARS) levels increased on arsenite exposure indicating arsenite induced hepatic oxidative stress. Kidney GSH, GSSG, catalase and TBARS remained unchanged on arsenite exposure. Treatment with DMSA was effective in increasing ALAD activity while, captopril was ineffective when given alone. Captopril when co-administered with DMSA also provided no additional beneficial effect on blood ALAD activity but significant brought altered platelet counts back to the normal value. In contrast, administration of captopril alone provided significant beneficial effects on hepatic oxidative stress, and in combination with DMSA provided a more pronounced recovery in the TBARS level compared to the individual effect of DMSA and captopril. Renal biochemical variables remained insensitive to arsenite and any of the treatments. Interestingly, combined administration of captopril with DMSA had a remarkable effect in depleting total arsenic concentration from blood and soft tissues. These results lead us to conclude that captopril administration during chelation treatment had some beneficial effects particularly on the protection of inhibited blood ALAD activity, and depletion of arsenic level. The study supports our earlier conclusion that a co-administration of an antioxidant is more beneficial than monotherapy with the chelating agents, in order to achieve optimal effects of chelation in arsenite toxicity.     

Cellular dynamics and tissue interactions of the dura mater during head development

During development and growth of the neurocranium, the dura mater regulates events in the underlying brain and overlying skull by the release of soluble factors and cellular activity. Morphogenesis of the cranial bones and sutures is dependent on tissue interactions with the dura mater, which control the size and shape of bones as well as sutural patency. Development of the brain also involves interactions with dura mater: secretion of stromal derived factor 1 (SDF-1) is a critical event in directing migration of the external granular layer precursors of the cerebellar cortex and the Cajal-Retzius (CR) cells of the cerebral cortex. The dura mater is also required for growth of the hippocampal dentate gyrus. Wnt1Cre/R26R transgenic reporter mice were used to study the origin and fates of the cells of dura mater during head development. The dura mater of mammals is derived entirely from the cranial neural crest. Beginning around neonatal day 10 (N 10), the dura mater is infiltrated by cells derived from paraxial mesoderm, which later come to predominate. Over the course of infancy, the neural crest-derived cells of the dura mater become sequestered in niche-like distribution characteristic of stem cells. Simultaneously, dura mater cells underlying the sagittal suture migrate upward into the mesodermally-derived mesenchyme separating the parietal bones. Although initially the parietal bones are formed entirely from paraxial mesoderm, the cellular composition gradually becomes chimeric and is populated mainly by neural crest-derived cells by N 30. This occurs as a consequence of osteoblastic differentiation at the dura mater interface and intravasation of neural crest-derived osteoclastic and other hematopoietic precursors. The isolated cells of the dura mater are multipotent in vitro, giving rise to osteoblasts, neuronal cells and other derivatives characteristic of cranial neural crest, possibly reflecting the multipotent nature of dura mater cells in vivo.     

Nonenzymatic synthesis of delta-aminolevulinate (ALA) by cobalt (Co++)

Cobalt, a metal known to modulate the heme biosynthetic enzymes, is shown to be capable of catalysing the formation of ALA through a transamination reaction. The transamination reaction follows a double-displacement reaction kinetics. Further, it has also been shown that the product of the reaction catalysed by cobalt can be used by the enzyme ALA dehydratase as the substrate in the formation of porphobilinogen. The formation of ALA by cobalt can be inhibited by the intermediates of the heme biosynthetic pathway, mainly protoporphyrin. Heme, on the other hand, does not have any effect on the reaction at all concentrations tested.     

Mitochondrial DNA-RFLP Analysis Distinguishes New CMS Sources in Pearl Millet (Pennisetum glaucum (L.) R. Br.)

Restriction fragment length polymorphism (RFLP) of mitochondrial (mt) DNA provides a rapid and effective method to assess heterogeneity among male sterile cytoplasms. Six isonuclear A-lines (81 A₁, with Tift 23A₁, cytoplasm, ICMA 88001 (= 81A_v) with Violaceum cytoplasm, 81A (=81A₄) with monodli = violaceum cytoplasm, Pb 310A₂ and Pb 311A₂ with A₂ cytoplasm from L 66A, and Pb 406A₃ with A₃ cytoplasm from L 67A), nine cytoplasmic male-sterility sources from Large-Seeded Genepool (LSGP 6, LSGP 14, LSGP 17, LSGP 22, LSGP 28, LSGP 36, LSGP 43, LSGP 55 and LSGP 66) and two each from Early Genepool (EGP 33 and EGP 15) and Population Varieties (PV 1 and PV 2) were characterized for variation in their mitochondrial genomes following Southern blot hybridizations using homologous (pearl millet 13.6 kb, 10.9 kb, 9.7 kb and 4.7 kb clones) and heterologous (maize atp6 and coxl clones) mitochondrial DNA (mtDNA) probes. Following cluster analysis based on similarity indices for the RFLP banding patterns observed, we identified seven cytoplasmic groups within LSGP. Two (LSGP 43 and LSGP 66) of these were quite distinct from each other as well as from other cytoplasms. This clearly indicates that besides serving as a source of diversity for agronomic and adaptation traits, broad-based gene pools can also provide diverse sources of cytoplasmic male sterility. These new CMS sources were also compared with standard CMS systems and cytoplasm-specific restriction fragments were identified.     

The transition zone in Hirschsprung's bowel contains abnormal hybrid ganglia with characteristics of extrinsic nerves

The aganglionic bowel in short-segment Hirschsprung's disease is characterized both by the absence of enteric ganglia and the presence of extrinsic thickened nerve bundles (TNBs). The relationship between the TNBs and the loss of enteric ganglia is unknown. Previous studies have described decreasing numbers of ganglia with increasing density of TNBs within the transition zone (TZ) between ganglionic and aganglionic gut, and there is some evidence of spatial contact between them in this region. To determine the cellular interactions involved, we have analysed the expression of perineurial markers of TNBs and enteric ganglionic markers for both neural cells and their ensheathing telocytes across four cranio-caudal segments consisting of most proximal ganglionic to most distal aganglionic from pull-through resected colon. We show that in the TZ, enteric ganglia are abnormal, being surrounded by perineurium cells characteristic of TNBs. Furthermore, short processes of ganglionic neurons extend caudally towards the aganglionic region, where telocytes in the TNB are located between the perineurium and nerve fibres into which they project telopodes. Thus, enteric ganglia within the TZ have abnormal structural characteristics, the cellular relationships of which are shared by the TNBs. These findings will help towards elucidation of the cellular mechanisms involved in the aetiology of Hirschsprung's disease.