Wnt signaling recruits KIF2A to the spindle to ensure chromosome congression and alignment during mitosis

Canonical Wnt signaling plays critical roles in development and tissue renewal by regulating β-catenin target genes. Recent evidence showed that β-catenin–independent Wnt signaling is also required for faithful execution of mitosis. However, the targets and specific functions of mitotic Wnt signaling still remain uncharacterized. Using phosphoproteomics, we identified that Wnt signaling regulates the microtubule depolymerase KIF2A during mitosis. We found that Dishevelled recruits KIF2A via its N-terminal and motor domains, which is further promoted upon LRP6 signalosome formation during cell division. We show that Wnt signaling modulates KIF2A interaction with PLK1, which is critical for KIF2A localization at the spindle. Accordingly, inhibition of basal Wnt signaling leads to chromosome misalignment in somatic cells and pluripotent stem cells. We propose that Wnt signaling monitors KIF2A activity at the spindle poles during mitosis to ensure timely chromosome alignment. Our findings highlight a function of Wnt signaling during cell division, which could have important implications for genome maintenance, notably in stem cells.

The canonical Wnt signaling pathway plays essential roles in embryonic development and tissue homeostasis (1, 2). In particular, Wnt signaling governs stem cell maintenance and proliferation in many tissues, and its misregulation is a common cause of tumor initiation (3, 4).Wnt ligands bind Frizzled (FZD) receptors and the coreceptors low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) (5). The activated receptor complexes cluster on Dishevelled (DVL) platforms and are internalized via caveolin into endosomes termed LRP6 signalosomes, which triggers sequential phosphorylation of LRP6 by GSK3β and CK1γ (6–10). LRP6 signalosomes recruit the β-catenin destruction complex, which contains the scaffold proteins AXIN1 and adenomatous polyposis coli, the kinases CK1α and GSK3β, and the E3 ubiquitin ligase β-TrCP (11). This recruitment inhibits GSK3β and releases β-TrCP, which leads to β-catenin stabilization and nuclear translocation in a IFT-A/KIF3A–dependent manner (12–16). LRP6 signalosomes mature into multivesicular bodies, sequestering the Wnt receptors together with GSK3β, thereby maintaining long-term activation of the Wnt pathway and promoting macropinocytosis (14, 17–21). In contrast to Wnt ligands, the Wnt inhibitor Dickkopf-related protein 1 (DKK1) induces the clathrin-dependent internalization and turnover of LRP5/6 and thereby abrogates canonical Wnt signaling (22).LRP6 signalosome formation peaks in mitosis (23, 24). On the one hand, the LRP6 competence to respond to Wnt ligands is promoted during G2/M by a priming phosphorylation at its intracellular domain by CDK14/16 and CCNY/CCNYL1 (24, 25). On the other hand, CDK1 phosphorylates and recruits B-cell CLL/lymphoma 9 (BCL9) to the mitotic LRP6 signalosomes (23). BCL9 protects the signalosome from clathrin-dependent turnover, thereby sustaining basal Wnt activity on the onset of mitosis.Mitotic Wnt signaling not only modulates β-catenin (24) but increasing evidence suggests that it promotes a complex posttranslational program during mitosis (26). For instance, we have shown that mitotic Wnt signaling promotes stabilization of proteins (Wnt/STOP), which is required for cell growth and ensures chromosome segregation in somatic and embryonic cells (23, 26–31). Particularly, basal Wnt/STOP activity maintains proper microtubule plus-end polymerization rates during mitosis, and its misregulation leads to whole chromosome missegregation (31, 32). Furthermore, mitotic Wnt signaling controls the orientation of the spindle (33) and promotes asymmetric division in stem cells through components of the LRP6 signalosome (34). Accordingly, several Wnt components functionally associate with centrosomes, kinetochores, and the spindle during mitosis (25, 33, 35, 36). Consequently, both aberrant up-regulation or down-regulation of Wnt signaling have been associated with chromosome instability (CIN) (31, 32, 35, 37), which is a hallmark of cancer (38). Despite the importance of these processes for tissue renewal and genome maintenance, the targets and specific functions of mitotic Wnt signaling remain largely uncharacterized.Kinesin family member 2A (KIF2A) is a member of the kinesin-13 group (KIF2A,B,C) of minus-end microtubule depolymerases (39–41). KIF2A is essential for the scaling of the spindle during early development (42) and plays critical roles in neurogenesis by modulating both cilium disassembly and neuronal wiring (43–47). In dividing cells, KIF2A was thought to be required for the assembly of a bipolar spindle due to a small interfering RNA (siRNA) off-target effect (48, 49). Current evidence supports a role of KIF2A in microtubule depolymerization at the spindle poles, which can generate pulling forces on attached kinetochores, thereby ensuring the congression, alignment, and segregation of chromosomes (50–56). Genetic depletion of KIF2A in mouse leads to neonatal lethality and to severe brain malformations, including microcephaly (43, 44, 57). KIF2A recruitment to microtubules is tightly coordinated by several protein kinases (45, 47, 50–52, 58–60). For instance, phosphorylation of KIF2A at several sites by Polo-like kinase 1 (PLK1) stimulates its recruitment to and activity at the spindle (45, 58, 61). On the other hand, Aurora kinase A and B inhibit KIF2A activity and restrict its subcellular localization during mitosis (50, 58, 60).Here, we show that mitotic Wnt signaling promotes chromosome congression and alignment in prometaphase by recruiting KIF2A to the spindle in both somatic cells and pluripotent stem cells. We found that KIF2A is recruited by the LRP6 signalosome during mitosis. Mechanistically, we identified that KIF2A clusters with DVL via the N-terminal and motor domains of the depolymerase. We show that Wnt signaling controls KIF2A interaction with PLK1, which is critical for KIF2A localization at the spindle poles. We propose that basal Wnt signaling ensures timely chromosome congression and alignment prior cell division by modulating the spindle minus-end depolymerization dynamics through KIF2A.     

Docosahexaenoic acid-enriched fish oil consumption modulates immunoglobulin responses to and clearance of enteric reovirus infection in mice

We hypothesized that consumption of the (n-3) PUFA, docosahexaenoic acid (DHA), modulates the mucosal immune response to enteric infection with respiratory enteric orphan virus (reovirus), a model intestinal pathogen. Mice were fed either AIN-93G control diet, containing 10 g/kg corn oil and 60 g/kg high oleic acid safflower oil, or AIN-93G, containing 10 g/kg corn oil and 60 g/kg DHA-enriched fish oil, for 4 wk and then orally gavaged with reovirus strain Type 1 Lang, (T1/L). Reovirus-specific IgA antibody was first detectable in the feces of mice fed a control diet at 6 d postinfection (PI) and was further elevated at 8 and 10 d PI. IgA responses in DHA-fed mice were similar at 6 and 8 d PI but greater at 10 d PI (P < 0.05). Both reovirus-specific serum IgA and IgG(2a) were comparably induced in mice fed control or DHA diets. Reovirus-specific IgA and IgG(2a) secretion by ex vivo Peyer's patch, lamina propria, and spleen cultures derived from control and DHA groups were comparable. Although both groups carried similar numbers of reovirus plaque forming units per intestine, DHA-fed mice shed nearly 10 times more viral RNA in feces than control mice at 2, 4, and 6 d PI (P < 0.05). However, viral RNA was not detectable in either group at 8 and 10 d. Taken together, these data suggest that DHA consumption did not markedly alter mucosal or systemic Ig responses to reovirus but delayed clearance of the virus from the intestinal tract.     

Contacts between medial and lateral perforant pathway fibers and parvalbumin expressing neurons in the subiculum of the rat

The entorhinal cortex (EC) projects via the perforant pathway to all subfields in the hippocampal formation. One can distinguish medial and lateral components in the pathway, originating in corresponding medial and lateral subdivisions of EC. We analyzed the innervation by medial and lateral perforant pathway fibers of parvalbumin-expressing neurons in the subiculum. A neuroanatomical tracer (biotinylated dextran amine, BDA) was stereotaxically injected in the medial or lateral entorhinal cortex, thus selectively labeling either perforant pathway component. Transport was allowed for 1 week. Transported BDA was detected with streptavidin-Alexa Fluor 488. Parvalbumin neurons were visualized via immunofluorescence histochemistry, using the fluorochrome Alexa Fluor 594. Via a random systematic sampling scheme using a two-channel, sequential-mode confocal laser scanning procedure, we obtained image series at high magnification from the molecular layer of the subiculum. Labeled entorhinal fibers and parvalbumin-expressing structures were three dimensionally (3D) reconstructed using computer software. Further computer analysis revealed that approximately 16% of the 3D objects ('boutons') of BDA-labeled fibers was engaged in contacts with parvalbumin-immunostained dendrites in the subiculum. Both medial and lateral perforant pathway fibers and their boutons formed such appositions. Contacts are suggestive for synapses. We found no significant differences between the medial and lateral components in the relative numbers of contacts. Thus, the medial and lateral subdivisions of the entorhinal cortex similarly tune the firing of principal neurons in the subiculum by way of parvalbumin positive interneurons in their respective terminal zones.     

Systemic diseases in age related cataract patients

Age related cataract is the most common cause of blindness in the world. Most of these patients are elderly and are likely to have various associated systemic diseases. Higher mortality has been reported in patients undergoing cataract surgery. In order to determine the prevalence of associated systemic disease, we carried out a large eye camp based study in 6103 age related cataract patients. Seventeen percent of our patients had systemic problems. Pulmonary disease was seen in 4.3%, cardiovascular disease and hypertension in 4.1%, diabetes mellitus in 3.8%, skin disorder in 1.4%, orodental disease requiring tooth extraction in 3%, and other diseases were seen in 0.4% of the cases. Seventy eight patients (1.27%) had significant systemic complications post-operatively, 46% of whom required hospitalization in a tertiary care center. Thus, all patients undergoing cataract surgery should be evaluated for associated systemic diseases to prevent morbidity and mortality in the preoperative, operative and postoperative period.     

The 18 kDa isoform of basic fibroblast growth factor is sufficient to stimulate human melanoma growth and angiogenesis

Basic fibroblast growth factor is the best-characterized autocrine growth factor in melanoma development and progression. We hypothesized that basic fibroblast growth factor might induce a more aggressive phenotype dependent on the amount of protein expressed in melanoma. Two human melanoma cell lines, M14 and 1F6, known to have low endogenous basic fibroblast growth factor expression and slow growth as subcutaneous xenografts, were stably transfected with vectors encoding either the 18 kDa or all (ALL) isoform proteins of human basic fibroblast growth factor. Different clones overexpressing the 18 kDa or ALL basic fibroblast growth factor proteins were easily obtained. Increased levels of basic fibroblast growth factor were secreted in conditioned medium and stored on the extracellular membrane. Biological activity of the overexpressed basic fibroblast growth factor was confirmed in a human umbilical vein endothelial cell proliferation assay. In 1F6 cells, overexpression of either 18 kDa or ALL basic fibroblast growth factor proteins resulted in up to two-fold shorter in-vitro doubling times (P<0.05). In addition, in vivo, both 18 kDa and ALL basic fibroblast growth factor-overexpressing 1F6 subcutaneous xenografts displayed significantly higher growth rates (P<0.05). In contrast, no major differences in in-vitro and in-vivo doubling times were observed when 18 kDa or ALL isoforms of basic fibroblast growth factor were overexpressed in M14 cells. Interestingly, basic fibroblast growth factor overexpression only affected the microvasculature in 1F6 xenografts. Although blood vessels in 1F6 parent tumors were large, 1F6 tumors overexpressing basic fibroblast growth factor contained numerous small, compressed vessels. Taken together, overexpression of the 18 kDa basic fibroblast growth factor protein only can promote autocrine melanoma cell growth and paracrine-driven angiogenesis.     

Expression and possible role of hPTTG1/securin in cutaneous malignant melanoma.

Human pituitary tumour-transforming gene 1 or hPTTG1 is a proto-oncogene that codes for securin, a protein involved in sister chromatid separation. Based on previous microarray data, we studied the expression of hPTTG1/securin in melanocytic lesions. In contrast to nevi and radial growth phase melanomas, securin was expressed by scattered cells in the vertical growth phase, suggesting a role in tumour progression. In a series of 29 nodular and 29 superficial spreading melanomas, matched for all histological prognostic parameters, securin expression was significantly correlated with the nodular subtype (P=0.018) and not related to thickness. In other cancers, hPTTG1 is involved in various oncogenic pathways, including induction of neovascularisation and aneuploidy, and inhibition of p53 activity. We found coexpression of securin with wild-type p53 in the same neoplastic cells in a minority of melanomas. Expression of securin was significantly correlated with the extent of aneuploidy but not with basic fibroblast growth factor immunoreactivity or microvessel density. DNA cytometry revealed that nuclei-overexpressing securin frequently showed tetraploidy or aneuploidy. Our data show that hPTTG1 is frequently overexpressed in nodular melanoma, and suggest that hPTTG1 may act as an oncogene in the vertical growth phase, either by inhibiting anaphase, thereby causing aneuploidy and genomic instability, or by modulating the function of p53, thereby impairing apoptosis.     

Surgical management of stab injuries of the heart

ITRODUCTION: Cardiac injuries today are not rare and they present a significant group of pathological cardiac diseases, with a large number of the injured (60-90%) dying before being transported to the hospital. CASE REPORT: A patient with a stab wound of the right chamber, upper abdomen and pelvis, in a state of hemorrhagic shock, was successfully managed after being reanimated. CONCLUSION: Good health service organization in the field and urgent transport of the injured should be obligatory in the management of heart trauma.     

Epidermal growth factor receptor (EGFR) and prostaglandin-endoperoxide synthase 2 (PTGS2) are prognostic biomarkers for patients with resected colorectal cancer liver metastases

Background:

Resection of colorectal cancer liver metastasis (CRCLM) with curative intent has long-term benefit in ∼40% of cases. Prognostic biomarkers are needed to improve clinical management and reduce futile surgeries. Expression of epidermal growth factor receptor (EGFR) and prostaglandin-endoperoxide synthase 2 (PTGS2; also known as cyclooxygenase-2) has been associated with carcinogenesis and survival. We investigated the prognostic value of EGFR and PTGS2 expression in patients with resected CRCLM.

Methods:

Formalin-fixed paraffin-embedded CRCLM tissue and corresponding primary tumour specimens from a multi-institutional cohort of patients who underwent liver resection between 1990 and 2010 were incorporated into tissue microarrays (TMAs). TMAs were stained for EGFR and PTGS2 by immunohistochemistry. The hazard rate ratio (HRR) for the association between expression in CRCLM and overall survival was calculated using a 500-fold cross-validation procedure.

Results:

EGFR and PTGS2 expression could be evaluated in 323 and 351 patients, respectively. EGFR expression in CRCLM was associated with poor prognosis (HRR 1.54; P<0.01) with a cross-validated HRR of 1.47 (P=0.03). PTGS2 expression was also associated with poor prognosis (HRR 1.60; P<0.01) with a cross-validated HRR of 1.63 (P<0.01). Expression of EGFR and PTGS2 remained prognostic after multivariate analysis with standard clinicopathological variables (cross-validated HRR 1.51; P=0.02 and cross-validated HRR 1.59; P=0.01, respectively). Stratification for the commonly applied systemic therapy regimens demonstrated prognostic value for EGFR and PTGS2 only in the subgroup of patients who were not treated with systemic therapy (HRR 1.78; P<0.01 and HRR 1.64; P=0.04, respectively), with worst prognosis when both EGFR and PTGS2 were highly expressed (HRR 3.08; P<0.01). Expression of PTGS2 in CRCLM was correlated to expression in patient-matched primary tumours (P=0.02, 69.2% concordance).

Conclusions:

EGFR and PTGS2 expressions are prognostic molecular biomarkers with added value to standard clinicopathological variables for patients with resectable CRCLM.     

Sniffing-related motor cortical potential: Topography and possible generators

This study estimated the whole-scalp topography and possible generators of the cortical potential associated with volitional self-paced inspirations (sniffs). In 17 healthy subjects we recorded a 32-channel electroencephalogram (EEG) during sniffing, for comparison during finger flexions. We averaged the EEG with respect to movement onset, and performed current source density and principal component analysis on the grand averaged data. We identified an early negative sniffing-related cortical potential starting ∼1.5 s before movement at the vertex, which, in its time-course and dipole orientation, closely resembled Bereitshaftspotential preceding finger flexions. Around the movement onset, its topography became unique with three negative current sources: one at the vertex, and two bilaterally over the fronto-temporal derivations. We conclude that sequential cortical activation in preparation for sniffing is similar to other volitional movements. The current sources at sniff onset at the vertex likely reflect somatotopic motor representation of the diaphragm, neck and intercostal muscles, whereas current sources over fronto-temporal derivations likely reflect the somatotopic representation of the orofacial muscles.