It has emerged recently that exosomes are potential carriers of pro-tumorigenic factors that participate in oncogenesis. However whether oncogenic transcription factors are transduced by exosomes is unknown. Hypoxia-inducible factor-1alpha (HIF-1α) transcriptionally regulates numerous key aspects of tumor development and progression by promoting a more aggressive tumor phenotype, characterized by increased proliferation and invasiveness coupled with neoangiogenesis. It has been shown that the principal oncoprotein of Epstein-Barr Virus (EBV), Latent Membrane Protein 1 (LMP1), drives oncogenic processes and tumor progression of the highly invasive EBV malignancy, nasopharyngeal carcinoma (NPC). We now demonstrate that endogenous HIF-1α is detectable in exosomes, and that LMP1 significantly increases levels of HIF-1α in exosomes. HIF-1 recovered from exosomes retains DNA-binding activity and is transcriptionally active in recipient cells after exosome uptake. We show also that treatment of EBV-negative cells with LMP1-exosomes increases migration and invasiveness of NP cell lines in functional assays, which correlates with the phenotype associated with Epithelial Mesenchymal Transition (EMT). In addition we provide evidence that HIF1α itself participates in exosome-mediated pro-metastatic effects in recipient cells, since exosome-mediated delivery of active and inactive forms of HIF-1α results in reciprocal changes in expression of E- and N-cadherins associated with EMT. Further, immunohistochemical analysis of NPC tumor tissues revealed direct correlation between protein levels of LMP1 and of the endosome/exosome marker tetraspanin, CD63, which suggests an increase in exosome formation in this EBV-positive malignancy. We hypothesize that exosome-mediated transfer of functional pro-metastatic factors by LMP1-positive NPC cells to surrounding tumor cells promotes cancer progression.
. Moreover, LMP1 induces HIF-1 DNA binding activity and upregulates HRE and VEGF promoter transcriptional activity. Finally, LMP1 increases the appearance of VEGF protein in extracellular fluids; induction of VEGF is suppressed by PD98059 or catalase. These results suggest that LMP1 increases HIF-1 activity through induction of HIF-1␣ protein expression, which is controlled by p42/p44 MAPK activity and H 2 O 2 . The ability of EBV, and specifically its major oncoprotein, LMP1, to induce HIF-1␣ along with other invasiveness and angiogenic factors reported previously discloses additional oncogenic properties of this tumor virus.
Nasopharyngeal carcinoma (NPC), an EBV-associated malignancy, is highly metastatic compared with other head and neck tumors, perhaps because of its viral link. Here, we show that the principal EBV oncoprotein, latent membrane protein 1 (LMP1), induces epithelial-mesenchymal transition (EMT) via Twist, a master transcriptional regulator in embryogenesis and newly implicated in metastasis, which, in turn, are likely to contribute to the highly metastatic character of NPC. LMP1 could induce EMT and its associated cell motility and invasiveness in a cell culture model, whereas expression of Twist small interfering RNA reversed LMP1-induced EMT. In diverse EBV-infected cell lines, expression of Twist correlates with expression of LMP1. Dominant-negative LMP1 could suppress Twist expression in EBV-positive cells, whereas LMP1 could induce Twist in EBV-negative nasopharyngeal cells. LMP1 signals through the nuclear factor-KB pathway, and an IKB superrepressor inhibited induction of Twist by LMP1. Finally, in human NPC tissues, expression of Twist and LMP1 is directly correlated and expression of Twist is associated with metastasis clinically. These results suggest that induction of Twist by a human viral oncoprotein LMP1 directly contributes to the metastatic nature of NPC. [Cancer Res 2007;67(5):1970-8]
Fast inhibition in the cortex is gated primarily at GABAergic synapses formed by local interneurons onto postsynaptic targets. Although GABAergic inputs to the somata and axon initial segments of neocortical pyramidal neurons are associated with direct inhibition of action potential generation, the role of GABAergic inputs to distal dendritic segments, including spines, is less well characterized. Because a significant proportion of inhibitory input occurs on distal dendrites and spines, it will be important to determine whether these GABAergic synapses are formed selectively by certain classes of presynaptic cells onto specific postsynaptic elements. By electron microscopic observations of synapses formed by different subtypes of nonpyramidal cells, we found that a surprisingly large fraction (33.4 Ϯ 9.3%) of terminals formed symmetrical synaptic junctions onto a subset of cortical spines that were mostly coinnervated by an asymmetrical terminal. Using VGLUT1 and VGLUT2 isoform of the glutamate vesicular transporter immunohistochemistry, we found that the double-innervated spines selectively received thalamocortical afferents expressing the VGLUT2 but almost never intracortical inputs expressing the VGLUT1. When comparing the volumes of differentially innervated spines and their synaptic junction areas, we found that spines innervated by VGLUT2-positive terminal were significantly larger than spines innervated by VGLUT1-positive terminal and that these spines had larger, and more often perforated, synapses than those of spines innervated by VGLUT1-positive afferent. These results demonstrate that inhibitory inputs to pyramidal cell spines may preferentially reduce thalamocortical rather than intracortical synaptic transmission and are therefore positioned to selectively gate extracortical information.
The purpose of the present work was to test the hypothesis that no more than one vesicle of transmitter can be liberated by an action potential at a single release site. Spontaneous and evoked IPSCs were recorded from interneurons in the molecular layer of cerebellar slices. Evoked IPSCs were obtained using either extracellular stimulation or paired recordings of presynaptic and postsynaptic neurons. Connections were identified as single-site synapses when evoked current amplitudes could be grouped into one peak that was well separated from the background noise. Peak amplitudes ranged from 30 to 298 pA. Reducing the release probability by lowering the external Ca2+ concentration or adding Cd2+ failed to reveal smaller quantal components. Some spontaneous IPSCs (1.4-2.4%) and IPSCs evoked at single-site synapses (2-6%) were followed within <5 msec by a secondary IPSC that could not be accounted for by random occurrence of background IPSCs. Nonlinear summation of closely timed events indicated that they involved activation of a common set of receptors and therefore that several vesicles could be released at the same release site by one action potential. An average receptor occupancy of 0.70 was calculated after single release events. At some single-site connections, two closely spaced amplitude peaks were resolved, presumably reflecting single and double vesicular release. Consistent with multivesicular release, kinetics of onset, decay, and latency were correlated to IPSC amplitude. We conclude that the one-site, one-vesicle hypothesis does not hold at interneuron-interneuron synapses.
Abstract. To understand the mechanisms of transport for organelles in the axon, we isolated and sequenced the cDNA encoding KIF4 from murine brain, and characterized the molecule biochemically and immunocytochemically. Complete amino acid sequence analysis of KIF4 and ultrastructural studies of KIF4 molecules expressed in Sf9 cells revealed that the protein contains 1,231 amino acid residues (Mr 139,550) and that the molecule (ll6-nm rod with globular heads and tail) consists of three domains: an NH2-terminal globular motor domain, a central u-helical stalk domain and a COOH-terminal tail domain. KIF4 protein has the property of nucleotide-dependent binding to microtubules, microtubule-activated ATPase activity, and microtubule plus-end-directed motility. Northern blot analysis and in situ hybridization demonstrated that KIF4 is strongly expressed in juvenile tissues including differentiated young neurons, while its expression is decreased considerably in adult mice except in spleen. Immunocytochemical studies revealed that KIF4 colocalized with membranous organelles both in growth cones of differentiated neurons and in the cytoplasm of cultured fibroblasts. During mitotic phase of cell cycle, KIF4 appears to colocalize with membranous organelles in the mitotic spindle. Hence we conclude that KIF4 is a novel microtubuleassociated anterograde motor protein for membranous organelles, the expression of which is regulated developmentally.
We have cloned two novel C-terminal motor domain-type kinesin superfamily motor proteins (KIFCs) from mouse brain by utilizing a KIFC-specific consensus sequence. The first protein was the murine homologue of CHO2 antigen, a member of the kar3-type mitotic motor subfamily, and we designated this protein KIFC1. The other protein, KIFC2 (792 amino acids), is novel, with no significant similarity to known kinesin superfamily proteins (KIFs). KIFC2 was specifically expressed in adult neurons, and was immunofluorescently localized to punctate structures in cell bodies and dendrites, but was not detected in axons. Electron microscopic analysis of the immunoisolated KIFC2-bound organelles revealed that KIFC2 associates with multivesicular body (mvb)-like organelles, suggesting that KIFC2 functions as the motor for the transport of mvb-like organelles in dendrites.
KIF (kinesin superfamily) proteins are microtubule-dependent molecular motors that play important roles in intracellular transport and cell division. The extent to which KIFs are involved in various transporting phenomena, as well as their regulation mechanism, are unknown. The identification of 16 new KIFs in this report doubles the existing number of KIFs known in the mouse. Conserved nucleotide sequences in the motor domain were amplified by PCR using cDNAs of mouse nervous tissue, kidney, and small intestine as templates. The new KIFs were studied with respect to their expression patterns in different tissues, chromosomal location, and molecular evolution. Our results suggest that ( i ) there is no apparent tendency among related subclasses of KIFs of cosegregation in chromosomal mapping, and ( ii ) according to their tissue distribution patterns, KIFs can be divided into two classes–i.e., ubiquitous and specific tissue-dominant. Further characterization of KIFs may elucidate unknown fundamental phenomena underlying intracellular transport. Finally, we propose a straightforward nomenclature system for the members of the mouse kinesin superfamily.
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