Loss of aPKCλ in Differentiated Neurons Disrupts the Polarity Complex but Does Not Induce Obvious Neuronal Loss or Disorientation in Mouse Brains

Cell polarity plays a critical role in neuronal differentiation during development of the central nervous system (CNS). Recent studies have established the significance of atypical protein kinase C (aPKC) and its interacting partners, which include PAR-3, PAR-6 and Lgl, in regulating cell polarization during neuronal differentiation. However, their roles in neuronal maintenance after CNS development remain unclear. Here we performed conditional deletion of aPKCλ, a major aPKC isoform in the brain, in differentiated neurons of mice by camk2a-cre or synapsinI-cre mediated gene targeting. We found significant reduction of aPKCλ and total aPKCs in the adult mouse brains. The aPKCλ deletion also reduced PAR-6β, possibly by its destabilization, whereas expression of other related proteins such as PAR-3 and Lgl-1 was unaffected. Biochemical analyses suggested that a significant fraction of aPKCλ formed a protein complex with PAR-6β and Lgl-1 in the brain lysates, which was disrupted by the aPKCλ deletion. Notably, the aPKCλ deletion mice did not show apparent cell loss/degeneration in the brain. In addition, neuronal orientation/distribution seemed to be unaffected. Thus, despite the polarity complex disruption, neuronal deletion of aPKCλ does not induce obvious cell loss or disorientation in mouse brains after cell differentiation.     

Platelets Promote Tumor Growth and Metastasis via Direct Interaction between Aggrus/Podoplanin and CLEC-2

The platelet aggregation-inducing factor Aggrus, also known as podoplanin, is frequently upregulated in several types of tumors and enhances hematogenous metastasis by interacting with and activating the platelet receptor CLEC-2. Thus, Aggrus–CLEC-2 binding could be a therapeutic molecular mechanism for cancer therapy. We generated a new anti-human Aggrus monoclonal antibody, MS-1, that suppressed Aggrus–CLEC-2 binding, Aggrus-induced platelet aggregation, and Aggrus-mediated tumor metastasis. Interestingly, the MS-1 monoclonal antibody attenuated the growth of Aggrus-positive tumors in vivo. Moreover, the humanized chimeric MS-1 antibody, ChMS-1, also exhibited strong antitumor activity against Aggrus-positive lung squamous cell carcinoma xenografted into NOD-SCID mice compromising antibody-dependent cellular cytotoxic and complement-dependent cytotoxic activities. Because Aggrus knockdown suppressed platelet-induced proliferation in vitro and tumor growth of the lung squamous cell carcinoma in vivo, Aggrus may be involved in not only tumor metastasis but also tumor growth by promoting platelet-tumor interaction, platelet activation, and secretion of platelet-derived factors in vivo. Our results indicate that molecular target drugs inhibiting specific platelet–tumor interactions can be developed as antitumor drugs that suppress both metastasis and proliferation of tumors such as lung squamous cell carcinoma.     

Point mutagenesis in mouse reveals contrasting pathogenetic effects between MEN2B- and Hirschsprung disease-associated missense mutations of the RET gene

Missense mutations of the RET gene have been identified in both multiple endocrine neoplasia (MEN) type 2A/B and Hirschsprung disease (HSCR: congenital absence of the enteric nervous system, ENS). Current consensus holds that MEN2A/B and HSCR are caused by activating and inactivating RET mutations, respectively. However, the biological significance of RET missense mutations in vivo has not been fully elucidated. In the present study, we introduced one MEN2B-associated (M918T) and two HSCR-associated (N394K and Y791F) RET missense mutations into the corresponding regions of the mouse Ret gene by genome editing (Ret^M919T, Ret^N396K and Ret^Y792F) and performed histological examinations of Ret-expressing tissues to understand the pathogenetic impact of each mutant in vivo. Ret^M919T/+ mice displayed MEN2B-related phenotypes, including C-cell hyperplasia and abnormal enlargement of the primary sympathetic ganglia. Similar sympathetic phenotype was observed in Ret^M919T/- mice, demonstrating a strong pathogenetic effect of the Ret M918T by a single-allele expression. In contrast, no abnormality was found in the ENS of mice harboring the Ret N394K or Y791F mutation. Most surprisingly, single-allele expression of RET N394K or Y791F was sufficient for normal ENS development, indicating that these RET mutants exert largely physiological function in vivo. This study reveals contrasting pathogenetic effects between MEN2B- and HSCR-associated RET missense mutations, and suggests that some of HSCR-associated RET missense mutations are by themselves neither inactivating nor pathogenetic and require involvement of other gene mutations for disease expressivity.     

Inhibitory synaptic transmission tuned by Ca2+ and glutamate through the control of GABAAR lateral diffusion dynamics

The GABAergic synapses, a primary inhibitory synapse in the mammalian brain, is important for the normal development of brain circuits, and for the regulation of the excitation-inhibition balance critical for brain function from the developmental stage throughout life. However, the molecular mechanism underlying the formation, maintenance, and modulation of GABAergic synapses is less understood compared to that of excitatory synapses. Quantum dot-single particle tracking (QD-SPT), a super-resolution imaging technique that enables the analysis of membrane molecule dynamics at single-molecule resolution, is a powerful tool to analyze the behavior of proteins and lipids on the plasma membrane. In this review, we summarize the recent application of QD-SPT in understanding of GABAergic synaptic transmission. Here we introduce QD-SPT experiments that provide further insights into the molecular mechanism supporting GABAergic synapses. QD-SPT studies revealed that glutamate and Ca²⁺ signaling is involved in (a) the maintenance of GABAergic synapses, (b) GABAergic long-term depression, and GABAergic long-term potentiation, by specifically activating signaling pathways unique to each phenomenon. We also introduce a novel Ca²⁺ imaging technique to describe the diversity of Ca²⁺ signals that may activate the downstream signaling pathways that induce specific biological output.     

Defective immune responses in mice lacking LUBAC‐mediated linear ubiquitination in B cells

The linear ubiquitin chain assembly complex (LUBAC) plays a crucial role in activating the canonical NF‐κB pathway, which is important for B‐cell development and function. Here, we describe a mouse model (B‐HOIP^Δlinear) in which the linear polyubiquitination activity of LUBAC is specifically ablated in B cells. Canonical NF‐κB and ERK activation, mediated by the tumour necrosis factor (TNF) receptor superfamily receptors CD40 and TACI, was impaired in B cells from B‐HOIP^Δlinear mice due to defective activation of the IKK complex; however, B‐cell receptor (BCR)‐mediated activation of the NF‐κB and ERK pathways was unaffected. B‐HOIP^Δlinear mice show impaired B1‐cell development and defective antibody responses to thymus‐dependent and thymus‐independent II antigens. Taken together, these data suggest that LUBAC‐mediated linear polyubiquitination is essential for B‐cell development and activation, possibly via canonical NF‐κB and ERK activation induced by the TNF receptor superfamily, but not by the BCR.     

Mother–calf interactions and social behavior development in Commerson’s dolphins (Cephalorhynchus commersonii)

Mother–calf interactions and the behaviors of mothers during separation from their calves were examined in four Commerson’s dolphin (Cephalorhynchus commersonii) mother–calf pairs. Four infants were observed: 56.8 h over 30 days from birth to 263 days of age, 36.9 h over 20 days from birth to 149 days of age, 10.4 h over 3 days from birth to 2 days of age, and 15.0 h over 3 days from birth to 2 days of age. All four pairs shared common characteristics in the rate and frequencies of mother–calf interactions and the behaviors of mothers during the first week of life. After the first week, individual differences in changes in the frequency of each behavior were observed. The three behaviors considered representative of maternal care (parallel swimming, synchronous breathing, and body-to-body contact) were frequently performed in the first week; thereafter, the frequencies declined. Separate behaviors of mothers were infrequent during the first week and increased with an increase in infants’ age. Bumping by infants increased with time, suggesting an increase in soliciting by calves and conflict between mothers and calves. The frequency of flipper-to-body rubbing also changed but in a complex manner, probably because the calves needed to learn how to perform this behavior from their mothers and because initiator and recipient of this behavior can be changed quickly.     

RNAi suppression of β-N-acetylglucosaminidase (BmFDL) for complex-type N-linked glycan synthesis in cultured silkworm cells

Glycoproteins have various biological functions including enzymatic activity, protein stability and others. Due to the presence of paucimannosidic N-linked glycans, recombinant proteins from an insect cell expression system may not be suitable for therapeutic use. Because baculovirus expression systems (BESs) are used to produce recombinant proteins, it is of interest to modify the endogenous N-glycosylation pathway in insects to mimic that of mammals. Using a soaking RNAi sensitive cell line, BmN4-SID1, has enabled us to suppress Bombyx mori FDL (BmFDL), an N-linked glycan-specific β-N-acetylglucosaminidase. Western blotting and MALDI-TOF MS demonstrated that the BmFDL depletion almost completely converted the paucimannosidic structures of the recombinant proteins produced by BES into a complex-type structure. This highly efficient, simple and low-cost method can be used for mass production of secretion proteins with complex-type N-linked glycans.     

Effects of Sphingomyelin Headgroup Size on Interactions with Ceramide

Sphingomyelins (SMs) and ceramides are known to interact favorably in bilayer membranes. Because ceramide lacks a headgroup that could shield its hydrophobic body from unfavorable interactions with water, accommodation of ceramide under the larger phosphocholine headgroup of SM could contribute to their favorable interactions. To elucidate the role of SM headgroup for SM/ceramide interactions, we explored the effects of reducing the size of the phosphocholine headgroup (removing one, two, or three methyls on the choline moiety, or the choline moiety itself). Using differential scanning calorimetry and fluorescence spectroscopy, we found that the size of the SM headgroup had no marked effect on the thermal stability of ordered domains formed by SM analog/palmitoyl ceramide (PCer) interactions. In more complex bilayers composed of a fluid glycerophospholipid, SM analog, and PCer, the thermal stability and molecular order of the laterally segregated gel domains were roughly identical despite variation in SM headgroup size. We suggest that that the association between PCer and SM analogs was stabilized by ceramide’s aversion for disordered phospholipids, by interfacial hydrogen bonding between PCer and the SM analogs, and by attractive van der Waals’ forces between saturated chains of PCer and SM analogs.     

Hyaluronan synthesis in cultured tobacco cells (BY‐2) expressing a chlorovirus enzyme: Cytological studies

Extraction of hyaluronan from animals or microbial fermentation has risks including contamination with pathogens and microbial toxins. In this work, tobacco cultured‐cells (BY‐2) were successfully transformed with a chloroviral hyaluronan synthase (cvHAS) gene to produce hyaluronan. Cytological studies revealed accumulation of HA on the cells, and also in subcellular fractions (protoplasts, miniplasts, vacuoplasts, and vacuoles). Transgenic BY‐2 cells harboring a vSPO‐cvHAS construct containing the vacuolar targeting signal of sporamin connected to the N‐terminus of cvHAS accumulated significant amounts of HA in vacuoles. These results suggested that cvHAS successfully functions on the vacuolar membrane and synthesizes/transports HA into vacuoles. Efficient synthesis of HA using this system provides a new method for practical production of HA. Biotechnol. Bioeng. 2013; 110: 1174–1179. © 2012 Wiley Periodicals, Inc.