A doublecortin containing microtubule-associated protein is implicated in mechanotransduction in Drosophila sensory cilia

Mechanoreceptors are sensory cells that transduce mechanical stimuli into electrical signals and mediate the perception of sound, touch and acceleration. Ciliated mechanoreceptors possess an elaborate microtubule cytoskeleton that facilitates the coupling of external forces to the transduction apparatus. In a screen for genes preferentially expressed in Drosophila campaniform mechanoreceptors, we identified DCX-EMAP, a unique member of the EMAP family (echinoderm–microtubule-associated proteins) that contains two doublecortin domains. DCX-EMAP localizes to the tubular body in campaniform receptors and to the ciliary dilation in chordotonal mechanoreceptors in Johnston's organ, the fly's auditory organ. Adult flies carrying a piggyBac insertion in the DCX-EMAP gene are uncoordinated and deaf and display loss of mechanosensory transduction and amplification. Electron microscopy of mutant sensilla reveals loss of electron-dense materials within the microtubule cytoskeleton in the tubular body and ciliary dilation. Our results establish a catalogue of candidate genes for Drosophila mechanosensation and show that one candidate, DCX-EMAP, is likely to be required for mechanosensory transduction and amplification

Novel CCL21-Vault Nanocapsule Intratumoral Delivery Inhibits Lung Cancer Growth

Based on our preclinical findings, we are assessing the efficacy of intratumoral injection of dendritic cells (DC) transduced with an adenoviral vector expressing the secondary lymphoid chemokine (CCL21) gene (Ad-CCL21-DC) in a phase I trial in advanced non-small cell lung cancer (NSCLC). While this approach shows immune enhancement, the preparation of autologous DC for CCL21 genetic modification is cumbersome, expensive and time consuming. We are evaluating a non-DC based approach which utilizes vault nanoparticles for intratumoral CCL21 delivery to mediate antitumor activity in lung cancer.Here we describe that vault nanocapsule platform for CCL21 delivery elicits antitumor activity with inhibition of lung cancer growth. Vault nanocapsule packaged CCL21 (CCL21-vaults) demonstrated functional activity in chemotactic and antigen presenting activity assays. Recombinant vaults impacted chemotactic migration of T cells and this effect was predominantly CCL21 dependent as CCL21 neutralization abrogated the CCL21 mediated enhancement in chemotaxis. Intratumoral administration of CCL21-vaults in mice bearing lung cancer enhanced leukocytic infiltrates (CXCR3(+)T, CCR7(+)T, IFNγ(+)T lymphocytes, DEC205(+) DC), inhibited lung cancer tumor growth and reduced the frequencies of immune suppressive cells [myeloid derived suppressor cells (MDSC), T regulatory cells (Treg), IL-10 T cells]. CCL21-vaults induced systemic antitumor responses by augmenting splenic T cell lytic activity against parental tumor cells.This study demonstrates that the vault nanocapsule can efficiently deliver CCL21 to sustain antitumor activity and inhibit lung cancer growth. The vault nanocapsule can serve as an "off the shelf" approach to deliver antitumor cytokines to treat a broad range of malignancies

Mutations in Eml1 lead to ectopic progenitors and neuronal heterotopia in mouse and human.

Neuronal migration disorders such as lissencephaly and subcortical band heterotopia are associated with epilepsy and intellectual disability. DCX, PAFAH1B1 and TUBA1A are mutated in these disorders; however, corresponding mouse mutants do not show heterotopic neurons in the neocortex. In contrast, spontaneously arisen HeCo mice display this phenotype, and our study revealed that misplaced apical progenitors contribute to heterotopia formation. While HeCo neurons migrated at the same speed as wild type, abnormally distributed dividing progenitors were found throughout the cortical wall from embryonic day 13. We identified Eml1, encoding a microtubule-associated protein, as the gene mutated in HeCo mice. Full-length transcripts were lacking as a result of a retrotransposon insertion in an intron. Eml1 knockdown mimicked the HeCo progenitor phenotype and reexpression rescued it. We further found EML1 to be mutated in ribbon-like heterotopia in humans. Our data link abnormal spindle orientations, ectopic progenitors and severe heterotopia in mouse and human

Xenopus Meiotic Microtubule-Associated Interactome

In metazoan oocytes the assembly of a microtubule-based spindle depends on the activity of a large number of accessory non-tubulin proteins, many of which remain unknown. In this work we isolated the microtubule-bound proteins from Xenopus eggs. Using mass spectrometry we identified 318 proteins, only 43 of which are known to bind microtubules. To integrate our results, we compiled for the first time a network of the meiotic microtubule-related interactome. The map reveals numerous interactions between spindle microtubules and the newly identified non-tubulin spindle components and highlights proteins absent from the mitotic spindle proteome. To validate newly identified spindle components, we expressed as GFP-fusions nine proteins identified by us and for first time demonstrated that Mgc68500, Loc398535, Nif3l1bp1/THOC7, LSM14A/RAP55A, TSGA14/CEP41, Mgc80361 and Mgc81475 are associated with spindles in egg extracts or in somatic cells. Furthermore, we showed that transfection of HeLa cells with siRNAs, corresponding to the human orthologue of Mgc81475 dramatically perturbs spindle formation in HeLa cells. These results show that our approach to the identification of the Xenopus microtubule-associated proteome yielded bona fide factors with a role in spindle assembly

Recruitment of the Major Vault Protein by InlK: A Listeria monocytogenes Strategy to Avoid Autophagy

L. monocytogenes is a facultative intracellular bacterium responsible for listeriosis. It is able to invade, survive and replicate in phagocytic and non-phagocytic cells. The infectious process at the cellular level has been extensively studied and many virulence factors have been identified. Yet, the role of InlK, a member of the internalin family specific to L. monocytogenes, remains unknown. Here, we first show using deletion analysis and in vivo infection, that InlK is a bona fide virulence factor, poorly expressed in vitro and well expressed in vivo, and that it is anchored to the bacterial surface by sortase A. We then demonstrate by a yeast two hybrid screen using InlK as a bait, validated by pulldown experiments and immunofluorescence analysis that intracytosolic bacteria via an interaction with the protein InlK interact with the Major Vault Protein (MVP), the main component of cytoplasmic ribonucleoproteic particules named vaults. Although vaults have been implicated in several cellular processes, their role has remained elusive. Our analysis demonstrates that MVP recruitment disguises intracytosolic bacteria from autophagic recognition, leading to an increased survival rate of InlK over-expressing bacteria compared to InlK− bacteria. Together these results reveal that MVP is hijacked by L. monocytogenes in order to counteract the autophagy process, a finding that could have major implications in deciphering the cellular role of vault particles

Expression and Cellular Distribution of Major Vault Protein: A Putative Marker for Pharmacoresistance in a Rat Model for Temporal Lobe Epilepsy

Summary: Purpose: Because drug transporters might play a role in the development of multidrug resistance (MDR), we investigated the expression of a vesicular drug transporter, the major vault protein (MVP), in a rat model for temporal lobe epilepsy. Methods: By using real-time polymerase chain reaction (PCR) analysis and immunocytochemistry, we quantified MVP mRNA and protein from the dentate gyrus (DG) and parahippocampal cortex (PHC) taken from EEG-monitored rats at 1 week after electrically induced status epilepticus (SE) and at 59 months after SE, when rats exhibit spontaneous seizures. Results: Within 1 week after SE, MVP mRNA levels increased in both DG and PHC compared with those in controls. In chronic epileptic rats, MVP mRNA was still significantly upregulated in the PHC, whereas in the DG, the expression returned to control levels. MVP protein increased within 1 day after SE in reactive microglial cells within most limbic regions; the hippocampus showed the highest expression at 1 week after SE. In chronic epileptic rats, MVP protein expression was largely decreased in most brain regions, but it was still high, especially in the piriform cortex. The occurrence of SE was a prerequisite for increased MVP expression, because no increase was found in electrically stimulated rats that did not exhibit SE. Conclusions: MVP expression is upregulated in chronic epileptic rats and may contribute to the development of pharmacoresistanc

Kinesin molecular motor Eg5 functions during polypeptide synthesis

The microtubule motor Eg5 is well known for its functions during mitosis. It is shown that during interphase, Eg5 associates with ribosomes and is required for efficient protein synthesis