Cell-to-cell progression of tobacco mosaic virus (TMV) infection in plants depends on virus-encoded movement protein (MP). Here we show that a conserved sequence motif in tobamovirus MPs shares similarity with a region in tubulins that is proposed to mediate lateral contacts between microtubule protofilaments. Point mutations in this motif confer temperature sensitivity to microtubule association and viral-RNA intercellular-transport functions of the protein, indicating that MP-interacting microtubules are functionally involved in the transport of vRNA to plasmodesmata. Moreover, we show that MP interacts with microtubule-nucleation sites. Together, our results indicate that MP may mimic tubulin assembly surfaces to propel vRNA transport by a dynamic process that is driven by microtubule polymerization.

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Teneurin-2, a vertebrate homologue of the Drosophila pairrule gene ten-m/odz, is revealed to be a membrane-bound transcription regulator. In the nucleus, the intracellular domain of teneurin-2 colocalizes with promyelocytic leukemia (PML) protein in nuclear bodies implicated in transcription control. Since Drosophila ten-m acts epistatically to another pair-rule gene opa, we investigated whether gene regulation by the mammalian opa homologue zic-1 was influenced by the intracellular domain of teneurin-2. We found that zic-mediated transcription from the apolipoprotein E promoter was inhibited. Release of the intracellular domain of teneurin-2 could be stimulated by homophilic interaction of the extracellular domain, and the intracellular domain was stabilized by proteasome inhibitors. We have previously shown that teneurin-2 is expressed by neurons belonging to the same functional circuit. Therefore, we hypothesize that homophilic interaction enables neurons to identify their targets and that the release of the intracellular domain of teneurin-2 provides them with a signal to switch their gene expression program from growth towards differentiation once the proper contact has been made.

SummaryThe movement protein (MP) of tobacco mosaic virus (TMV) is essential for spread of the viral RNA genome from cell to cell. During infection, the MP associates with microtubules, and it has been proposed that the cytoskeleton transports the viral ribonucleoprotein complex from ER sites of synthesis to plasmodesmata through which infection spreads into adjacent cells. However, microtubule association of MP was observed in cells undergoing late infection rather than in cells undergoing early infection at the leading edge of expanding infection sites where virus RNA cell-to-cell spread occurs. Therefore, alternative roles for microtubules in virus infection have been proposed, including a role in MP degradation. To further investigate the role of microtubules in virus pathogenesis, we tested the ef®ciency of cell-to-cell spread of infection and microtubule association of the MP in response to changes in temperature. We show that the subcellular distribution of MP is temperature-dependent and that a higher ef®ciency of intercellular transport of virus RNA at elevated temperatures corresponds to an increased association of MP with microtubules early in infection.

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Intercellular transport of tobacco mosaic virus (TMV) RNA involves the accumulation of virus-encoded movement protein (MP) in plasmodesmata (Pd), in endoplasmic reticulum (ER)-derived inclusion bodies, and on microtubules. The functional significance of these interactions in viral RNA (vRNA) movement was tested in planta and in protoplasts with TMV derivatives expressing N- and C-terminal deletion mutants of MP fused to the green fluorescent protein. Deletion of 55 amino acids from the C terminus of MP did not interfere with the vRNA transport function of MP:GFP but abolished its accumulation in inclusion bodies, indicating that accumulation of MP at these ER-derived sites is not a requirement for function in vRNA intercellular movement. Deletion of 66 amino acids from the C terminus of MP inactivated the protein, and viral infection occurred only upon complementation in plants transgenic for MP. The functional deficiency of the mutant protein correlated with its inability to associate with microtubules and, independently, with its absence from Pd at the leading edge of infection. Inactivation of MP by N-terminal deletions was correlated with the inability of the protein to target Pd throughout the infection site, whereas its associations with microtubules and inclusion bodies were unaffected. The observations support a role of MP-interacting microtubules in TMV RNA movement and indicate that MP targets microtubules and Pd by independent mechanisms. Moreover, accumulation of MP in Pd late in infection is insufficient to support viral movement, confirming that intercellular transport of vRNA relies on the presence of MP in Pd at the leading edge of infection.

The extracellular matrix protein tenascin-C (TNC) is up-regulated in processes influenced by mechanical stress, such as inflammation, tissue remodeling, wound healing, and tumorigenesis. Cyclic strain-induced TNC expression depends on RhoA-actin signaling, the pathway that regulates transcriptional activity of serum response factor (SRF) by its coactivator megakaryoblastic leukemia-1 (MKL1). Therefore, we tested whether MKL1 controls TNC transcription. We demonstrate that overexpression of MKL1 strongly induces TNC expression in mouse NIH3T3 fibroblasts and normal HC11 and transformed 4T1 mammary epithelial cells. Part of the induction was dependant on SRF and a newly identified atypical CArG box in the TNC promoter. Another part was independent of SRF but required the SAP domain of MKL1. An MKL1 mutant incapable of binding to SRF still strongly induced TNC, while induction of the SRF target c-fos was abolished. Cyclic strain failed to induce TNC in MKL1-deficient but not in SRF-deficient fibroblasts, and strain-induced TNC expression strongly depended on the SAP domain of MKL1. Promoter-reporter and chromatin immunoprecipitation experiments unraveled a SAP-dependent, SRF-independent interaction of MKL1 with the proximal promoter region of TNC, attributing for the first time a functional role to the SAP domain of MKL1 in regulating gene expression.

BackgroundThe main cause of death of breast cancer patients is not the primary tumor itself but the metastatic disease. Identifying breast cancer-specific signatures for metastasis and learning more about the nature of the genes involved in the metastatic process would 1) improve our understanding of the mechanisms of cancer progression and 2) reveal new therapeutic targets. Previous studies showed that the transcriptional regulator megakaryoblastic leukemia-1 (Mkl1) induces tenascin-C expression in normal and transformed mammary epithelial cells. Tenascin-C is known to be expressed in metastatic niches, is highly induced in cancer stroma and promotes breast cancer metastasis to the lung.MethodsUsing HC11 mammary epithelial cells overexpressing different Mkl1 constructs, we devised a subtractive transcript profiling screen to identify the mechanism by which Mkl1 induces a gene set co-regulated with tenascin-C. We performed computational analysis of the Mkl1 target genes and used cell biological experiments to confirm the effect of these gene products on cell behavior. To analyze whether this gene set is prognostic of accelerated cancer progression in human patients, we used the bioinformatics tool GOBO that allowed us to investigate a large breast tumor data set linked to patient data.ResultsWe discovered a breast cancer-specific set of genes including tenascin-C, which is regulated by Mkl1 in a SAP domain-dependent, serum response factor-independent manner and is strongly implicated in cell proliferation, cell motility and cancer. Downregulation of this set of transcripts by overexpression of Mkl1 lacking the SAP domain inhibited cell growth and cell migration. Many of these genes are direct Mkl1 targets since their promoter-reporter constructs were induced by Mkl1 in a SAP domain-dependent manner. Transcripts, most strongly reduced in the absence of the SAP domain were mechanoresponsive. Finally, expression of this gene set is associated with high-proliferative poor-outcome classes in human breast cancer and a strongly reduced survival rate for patients independent of tumor grade.ConclusionsThis study highlights a crucial role for the transcriptional regulator Mkl1 and its SAP domain during breast cancer progression. We identified a novel gene set that correlates with bad prognosis and thus may help in deciding the rigor of therapy.