T-Cell Receptor Microclusters Critical for T-Cell Activation Are Formed Independently of Lipid Raft Clustering

We studied the function of lipid rafts in generation and signaling of T-cell receptor microclusters (TCR-MCs) and central supramolecular activation clusters (cSMACs) at immunological synapse (IS). It has been suggested that lipid raft accumulation creates a platform for recruitment of signaling molecules upon T-cell activation. However, several lipid raft probes did not accumulate at TCR-MCs or cSMACs even with costimulation and the fluorescence resonance energy transfer (FRET) between TCR or LAT and lipid raft probes was not induced at TCR-MCs under the condition of positive induction of FRET between CD3ζ and ZAP-70. The analysis of LAT mutants revealed that raft association is essential for the membrane localization but dispensable for TCR-MC formation. Careful analysis of the accumulation of raft probes in the cell interface revealed that their accumulation occurred after cSMAC formation, probably due to membrane ruffling and/or endocytosis. These results suggest that lipid rafts control protein translocation to the membrane but are not involved in the clustering of raft-associated molecules and therefore that the lipid rafts do not serve as a platform for T-cell activation.Lipid rafts are specialized liquid-ordered membrane microdomains that are enriched in cholesterol and sphingolipids. Many studies using various methodologies have shown that lipid rafts exist as leaflets less than 200 nm in size and float on the plasma membrane (6, 10, 24, 28, 32). They have been implied to play a role in protein sorting and cell activation as a platform by recruiting various signaling molecules such as Src family kinases, G proteins, and adaptor molecules. Because of size limitation, all of the raft-associated molecules could not be accommodated on the same lipid raft, and heterogeneity of lipid rafts both in size and in the repertoire of resident molecules has been suggested (22). The functional importance of lipid rafts in signal transduction has been particularly appreciated in T-cell activation through the T-cell receptor (TCR). Some of the initial observations in this area included the findings that cross-linking of the raft-associated ganglioside GM1 induces T-cell activation (12) and that a mutant of LAT, a membrane adaptor protein, that was unable to localize to rafts failed to induce activation signals (33). Since then, increasing data have demonstrated that lipid raft accumulation creates a platform to stabilize the signaling complex for T-cell activation (13, 29).T cells are activated upon recognition of peptide-major histocompatibility complex (MHC) complexes expressed on antigen-presenting cells (APC). An immunological synapse (IS) is formed at the interface between the T cell and the APC where a specialized segregated structure of T-cell surface receptors is generated. This supramolecular activation cluster (SMAC) contains the TCR in the central region (cSMAC) and lymphocyte function-associated antigen 1 (LFA-1) in the peripheral region (pSMAC). The accumulation of lipid rafts at this interface, particularly in the cSMAC, has been suggested to create a transient structure to mediate signal transduction (13, 17). In addition, CD28-mediated costimulation has been suggested to enhance lipid raft accumulation and TCR activation (29). However, the idea that lipid rafts accumulated in the cSMAC serve as the platform for T-cell activation has been controversial; the accumulation of the lipid raft was only partial in the contact area (3), or the concentration of lipid raft was constant even in the area of T-cell activation (5, 8, 28, 32). These variations could be partly attributed to differences in experimental approaches such as the cell systems being analyzed, stimulation conditions, and detection methods, including imaging and biochemical fractionation. The idea that the cSMAC is the site responsible for inducing signals for T-cell activation has been recently revised based on analysis of the dynamic assembly of signaling complexes upon TCR stimulation. Analysis of T-cell activation using a planar membrane system containing glycosylphosphatidylinositol (GPI)-anchored MHC-peptide complexes and the LFA-1 ligand intercellular adhesion molecule 1 (ICAM-1) revealed that small clusters containing approximately a hundred TCRs, kinases, and adaptors, which we termed TCR microclusters (MCs), were generated at the initial contact sites. This was followed by translocation of the MCs to the center of the interface to generate a cSMAC (31). Since protein phosphorylation, including that of ZAP-70, was induced in the TCR-MCs and Ca²⁺ mobilization was induced in parallel with the formation of TCR-MCs, these MCs appear to be the very first and minimum unit for generating TCR activation signals (31). Furthermore, a major costimulatory receptor, CD28, forms clusters which are also colocalized in TCR-MCs to regulate costimulatory signals (30).Among these TCR proximal signaling molecules, LAT is a well-studied raft-associated membrane adaptor protein that is indispensable for TCR activation. LAT is phosphorylated by ZAP-70 and then behaves as a signal scaffold, recruiting various signaling adaptors and effector molecules such as phospholipase Cγ (PLCγ), SLP-76, and Grb2/Gads. Because mutation of LAT palmitoylation sites (C26,29A) resulted in its dislocation from lipid rafts and defective signaling, it was concluded that the association with lipid rafts is essential for the function of LAT (33). However, a recent study showed that this mutant LAT has impaired trafficking to the plasma membrane in the Jurkat T-cell line (27), raising the question of whether the impaired signaling resulting from this LAT mutation was due to dislocation from the raft or defective trafficking to the membrane.Here, we analyzed the role of lipid rafts in T-cell activation, particularly their relationship with immunological synapse formation (9). Provided that lipid raft functions as a platform for T-cell activation, the new idea that TCR-MCs serve as the signal unit for activation would predict that lipid raft could be accumulated in or interact with TCR-MCs (29).Utilizing several lipid raft probes, which retain the capability of raft localization but lack signaling capacity, we found that the full-length LAT generated MCs, but none of the raft probes formed visible clusters at TCR-MCs or cSMAC, even in conjunction with CD28-mediated costimulation. Furthermore, no significant interaction between lipid rafts and TCR-MCs was revealed by fluorescence resonance energy transfer (FRET) analysis. Conversely, the non-raft-localizing LAT mutant showed MC formation upon TCR stimulation. These results suggest that lipid rafts do not serve as a platform for TCR signaling but rather regulate the traffic/recruitment of proteins to the plasma membrane. Furthermore, our data indicate that the previous observation of lipid raft accumulation at the cSMAC may reflect membrane ruffling and endocytosis rather than active formation of signal platform.     

Rate-dependent shortening of action potential duration increases ventricular vulnerability in failing rabbit heart

Congestive heart failure (CHF) predisposes to ventricular fibrillation (VF) in association with electrical remodeling of the ventricle. However, much remains unknown about the rate-dependent electrophysiological properties in a failing heart. Action potential properties in the left ventricular subepicardial muscles during dynamic pacing were examined with optical mapping in pacing-induced CHF (n=18) and control (n=17) rabbit hearts perfused in vitro. Action potential durations (APDs) in CHF were significantly longer than those observed for controls at basic cycle lengths (BCLs)>1,000 ms but significantly shorter at BCLs<400 ms. Spatial APD dispersions were significantly increased in CHF versus control (by 17-81%), and conduction velocity was significantly decreased in CHF (by 6-20%). In both groups, high-frequency stimulation (BCLs<150 ms) always caused spatial APD alternans; spatially concordant alternans and spatially discordant alternans (SDA) were induced at 60% and 40% in control, respectively, whereas 18% and 82% in CHF. SDA in CHF caused wavebreaks followed by reentrant excitations, giving rise to VF. Incidence of ventricular tachycardia/VFs elicited by high-frequency dynamic pacing (BCLs<150 ms) was significantly higher in CHF versus control (93% vs. 20%). In CHF, left ventricular subepicardial muscles show significant APD shortenings at short BCLs favoring reentry formations following wavebreaks in association with SDA. High-frequency excitation itself may increase the vulnerability to VF in CHF.     

HpSumf1 is involved in the activation of sulfatases responsible for regulation of skeletogenesis during sea urchin development

Sulfatases such as arylsulfatase and heparan sulfate 6-O-endosulfatase play important roles in morphogenesis during sea urchin development. For the activation of these sulfatases, Cα-formylglycine formation by sulfatase modifying factor (Sumf) is required. In this study, to clarify the regulatory mechanisms for the activation of sulfatases during sea urchin development, we examined the expression and function of the Hemicentrotus pulcherrimus homologs of Sumf1 and Sumf2 (HpSumf1 and HpSumf2, respectively). Expression of HpSumf1 but not HpSumf2 mRNA was dynamically changed during early development. Functional analyses of recombinant HpSumf1 and HpSumf2 using HEK293T cells expressing mouse arylsulfatase A (ArsA) indicated that HpSumf1 and HpSumf2 were both able to activate mammalian ArsA. Knockdown of HpSumf1 using morpholino antisense oligonucleotides caused abnormal spicule formation in the sea urchin embryo. Injection of HpSumf2 mRNA had no effect on skeletogenesis, while injection of HpSumf1 mRNA induced severe supernumerary spicule formation. Taken together, these findings suggest that HpSumf1 is involved in the activation of sulfatases required for control of skeletogenesis.     

Development of a sensitive screening method for selecting monoclonal antibodies to be internalized by cells

Antibody–drug conjugates (ADCs), drugs developed by conjugation of an anticancer agent to a monoclonal antibody (mAb), have lately attracted attention in cancer therapy because ADCs can directly bind cancer cells and kill them. Although mAbs for ADCs must be internalized by the target cells, few methods are available for screening mAbs for their ability to be internalized by cells. We have developed a recombinant protein, termed DT3C, which consists of diphtheria toxin (DT) lacking the receptor-binding domain but containing the C1, C2, and C3 domains of Streptococcus protein G (3C). When a mAb–DT3C conjugate, which functions in vitro like an ADC, reduces the viability of cancer cells, the mAb being tested must have been internalized by the target cells. DT3C can thus be a tool to identify efficiently and easily mAbs that can be internalized by cells, thereby enhancing the development of promising ADCs.     

Sterol Side Chain Reductase 2 Is a Key Enzyme in the Biosynthesis of Cholesterol,the Common Precursor of Toxic Steroidal Glycoalkaloids in Potato

Potatoes (Solanum tuberosum) contain α-solanine and α-chaconine, two well-known toxic steroidal glycoalkaloids (SGAs). Sprouts and green tubers accumulate especially high levels of SGAs. Although SGAs were proposed to be biosynthesized from cholesterol, the biosynthetic pathway for plant cholesterol is poorly understood. Here, we identify sterol side chain reductase 2 (SSR2) from potato as a key enzyme in the biosynthesis of cholesterol and related SGAs. Using in vitro enzyme activity assays, we determined that potato SSR2 (St SSR2) reduces desmosterol and cycloartenol to cholesterol and cycloartanol, respectively. These reduction steps are branch points in the biosynthetic pathways between C-24 alkylsterols and cholesterol in potato. Similar enzymatic results were also obtained from tomato SSR2. St SSR2-silenced potatoes or St SSR2-disrupted potato generated by targeted genome editing had significantly lower levels of cholesterol and SGAs without affecting plant growth. Our results suggest that St SSR2 is a promising target gene for breeding potatoes with low SGA levels.     

Characterization of vascular endothelial growth factor (VEGF) in the uterine cervix over pregnancy: effects of denervation and implications for cervical ripening.

Bilateral neurectomy of the pelvic nerve (BLPN) that carries uterine cervix-related sensory nerves induces dystocia, and administration of its vasoactive neuropeptides induces changes in the cervical microvasculature, resembling those that occur in the ripening cervix. This study was designed to test the hypothesis that (a) the cervix of pregnant rats expresses vascular endothelial growth factor (VEGF) and components of the angiogenic signaling pathway [VEGF receptors (Flt-1, KDR), activity of protein kinase B, Akt (phosphorylated Akt), and endothelial nitric oxide synthase (eNOS)] and von Willebrand Factor (vWF) and that these molecules undergo changes with pregnancy, and (b) bilateral pelvic neurectomy (BLPN) alters levels of VEGF concentration in the cervix. Using RT-PCR and sequencing, two VEGF isoforms, 120 and 164, were identified in the rat cervix. VEGF, VEGF receptor-1 (Flt-1), eNOS, and vWF immunoreactivities (ir) were localized in the microvasculature of cervical stroma. Their protein levels increased during pregnancy but decreased to control levels by 2 days postpartum. VEGF receptor-2 (KDR)-ir was confined to the epithelium of the endocervix. BLPN downregulated levels of VEGF by a third. Therefore, the components of the angiogenic signaling pathway are expressed in the cervix and change over pregnancy. Furthermore, angiogenic and sensory neuronal factors may be important in regulating the dynamic microvasculature in the ripening cervix and may subsequently play a role in cervical ripening and the birth process.