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Post-translational modifications (PTM) of proteins can control complex and dynamic cellular processes via regulating interactions between key proteins. To understand these regulatory mechanisms, it is critical that we can profile the PTM-dependent protein–protein interactions. However, identifying these interactions can be very difficult using available approaches, as PTMs can be dynamic and often mediate relatively weak protein–protein interactions. We have recently developed CLASPI (cross-linking-assisted and stable isotope labeling in cell culture-based protein identification), a chemical proteomics approach to examine protein–protein interactions mediated by methylation in human cell lysates. Here, we report three extensions of the CLASPI approach. First, we show that CLASPI can be used to analyze methylation-dependent protein–protein interactions in lysates of fission yeast, a genetically tractable model organism. For these studies, we examined trimethylated histone H3 lysine-9 (H3K9Me3)-dependent protein–protein interactions. Second, we demonstrate that CLASPI can be used to examine phosphorylation-dependent protein–protein interactions. In particular, we profile proteins recognizing phosphorylated histone H3 threonine-3 (H3T3-Phos), a mitotic histone “mark” appearing exclusively during cell division. Our approach identified survivin, the only known H3T3-Phos-binding protein, as well as other proteins, such as MCAK and KIF2A, that are likely to be involved in weak but selective interactions with this histone phosphorylation “mark”. Finally, we demonstrate that the CLASPI approach can be used to study the interplay between histone H3T3-Phos and trimethylation on the adjacent residue lysine 4 (H3K4Me3). Together, our findings indicate the CLASPI approach can be broadly applied to profile protein–protein interactions mediated by PTMs.

The stable propagation of genetic material during cell division depends on the congression of chromosomes to the spindle equator before the cell initiates anaphase. It is generally assumed that congression requires that chromosomes are connected to the opposite poles of the bipolar spindle (“bioriented”). In mammalian cells, we found that chromosomes can congress before becoming bioriented. By combining the use of reversible chemical inhibitors, live-cell light microscopy, and correlative electron microscopy, we found that monooriented chromosomes could glide toward the spindle equator alongside kinetochore fibers attached to other already bioriented chromosomes. This congression mechanism depended on the kinetochore-associated, plus end–directed microtubule motor CENP-E (kinesin-7).

Metaphase spindles assemble to a steady state in length by mechanisms that involve microtubule dynamics and motor proteins, but they are incompletely understood. We found that Xenopus extract spindles recapitulate the length of egg meiosis II spindles, by using mechanisms intrinsic to the spindle. To probe these mechanisms, we perturbed microtubule polymerization dynamics and opposed motor proteins and measured effects on spindle morphology and dynamics. Microtubules were stabilized by hexylene glycol and inhibition of the catastrophe factor mitotic centromere-associated kinesin (MCAK) (a kinesin 13, previously called XKCM) and destabilized by depolymerizing drugs. The opposed motors Eg5 and dynein were inhibited separately and together. Our results are consistent with important roles for polymerization dynamics in regulating spindle length, and for opposed motors in regulating the relative stability of bipolar versus monopolar organization. The response to microtubule destabilization suggests that an unidentified tensile element acts in parallel with these conventional factors, generating spindle shortening force.

We investigated the mechanism by which meiotic spindles become bipolar and the correlation between bipolarity and poleward flux, using Xenopus egg extracts. By speckle microscopy and computational alignment, we find that monopolar sperm asters do not show evidence for flux, partially contradicting previous work. We account for the discrepancy by describing spontaneous bipolarization of sperm asters that was missed previously. During spontaneous bipolarization, onset of flux correlated with onset of bipolarity, implying that antiparallel microtubule organization may be required for flux. Using a probe for TPX2 in addition to tubulin, we describe two pathways that lead to spontaneous bipolarization, new pole assembly near chromatin, and pole splitting. By inhibiting the Ran pathway with excess importin-alpha, we establish a role for chromatin-derived, antiparallel overlap bundles in generating the sliding force for flux, and we examine these bundles by electron microscopy. Our results highlight the importance of two processes, chromatin-initiated microtubule nucleation, and sliding forces generated between antiparallel microtubules, in self-organization of spindle bipolarity and poleward flux.

A 46-year-old woman attended the gynaecology clinic complaining of right iliac fossa pain and dyspareunia of 18 months duration. Her symptoms were gradually worsening. She had a history of hysterectomy and left salpingoophorectomy for menorrhagia caused by fibroids. The patient had a body mass index of 29. Bimanual pelvic examination revealed right adnexal fullness which was confirmed on ultrasound scan to be an ovarian cyst. After appropriate counselling a right oophorectomy was planned. The procedure was performed through a transverse suprapubic incision. During the operation the abdominal wall was held apart with a self-retaining retractor. It was not necessary to open the retroperitoneal space because the ovary was lying adherent to the vaginal vault peritoneum. The procedure was uneventful. In the immediate postoperative period she complained of pain in the right anterior and medial aspect of the thigh. She also showed signs of significant intraperitoneal bleed and developed mild haematuria. Repeat exploratory laparotomy was performed to stop haemorrhage. On the fourth postoperative day she fell down when attempting to walk. She described pain and numbness in the right anterior thigh extending from the inguinal crease and distally to the knee and slightly below the knee on the lateral border. Following recovery from surgery a specialist neurological examination showed weakness of right hip flexion and knee extension, absence of the right knee jerk, and anaesthesia over the anterior and medial aspects of the right thigh. A right femoral neuropathy, presumed secondary to the surgical procedure, was diagnosed. Peripheral neurophysiological studies showed acute denervation of the right vastus lateralis with a marked loss of motor units confirming the clinical diagnosis. The patient started physiotherapy and was treated with amitryptiline and tramadol for her pain. After 8 months there was appreciable improvement in her symptoms. She was started on gabapentin and remains under follow up in a pain clinic.

This article 'Grain Loss Due to Rat Damage' appeared in the International Rice Research Newsletter series, created by the International Rice Research Institute (IRRI). The primary objective of this publication was to expedite communication among scientists concerned with the development of improved technology for rice and for rice based cropping systems. This publication will report what scientists are doing to increase the production of rice in as much as this crop feeds the most densely populated and land scarce nations in the world.